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they rarely have a fistulous communication with the respiratory or gastrointestinal tract , and thus cystic overinflation and infection are unlikely . to our knowledge , this is the first report of infradiaphragmatic retroperitoneal els complicated with infection .
a 34-yr - old male who presented with a 3-week history of epigastric discomfort and a 1-week history of fever was referred to our hospital .
he had been treated with intravenous antibiotics for 1 week at the other hospital . on admission
the patient 's body temperature was 37.7 and blood laboratory studies suggested an infectious condition ( wbc 15,700/l , esr 120 mm / hr , crp 63.2 mg / l , amylase 96
chest radiograph showed an elevated left hemidiaphragm and a calcified mass in the left upper abdomen and computed tomographic scanning revealed that a 16-cm , multiseptated , dumbbell - shaped , huge cystic tumor containing dense mural calcifications was located in the medial aspect to the left lobe of the liver and spleen , and in the upper anterior aspect of the left kidney .
the mass extended to the pancreatic tail and its apex was abutting the heart ( fig .
1 ) . on the following day , 850 ml of thick yellowish pus was drained by sonography - guided fine needle aspiration for the purpose of infection control and diagnosis , but no microscopic organisms were found in repeated culture studies . on the 8th day of admission ,
magnetic resonance imaging showed a much reduced cyst size , and patient 's body temperature was normalized but blood laboratory data revealed still infectious condition ( wbc 9,700/l , esr 120 mm / hr , crp 44.7
the clinical diagnosis was of a subphrenic abscess or pancreatic pseudocyst and surgical treatment was needed . on the 10th day ,
the procedure was performed with the patient in the left semi - lateral position with a small roll placed posterior to elevate the left side .
a thoracoabdominal incision was made through the 11th intercostal space and a careful inspection of the pleural and retroperitoneal spaces was performed .
no abnormal findings were revealed in the pleural space but a cystic mass with adhesion to the surrounding organs was found in the retroperitoneal space . calcified materials and thick yellowish pus
careful dissection of the adhesions between the cyst and the surrounding structures was performed and most of these pathologic lesions were removed but a severe adhesive lesion between the aorta and diaphragm beneath the heart could be not removed . and
histopathologically , the cyst was lined by ciliated pseudostratified columnar epithelium and surfactants secreted by type ii pneumocytes were observed by special staining ( fig .
3 ) . in the cystic contents , many wbc and necrotic materials were found but there were no microorganisms in culture study .
the patient was discharged 10 days after the operation and the remnant mass had nearly disappeared at a chest radiograph obtained 3 months later .
the etiology of infradiaphragmatic extralobar pulmonary sequestration has been suggested to be caused by the trapping of sequestered tissue within the infradiaphragmatic space just before pleuroperitoneal membrane closure between gestation weeks 5 and 8 ( 1 ) .
infradiaphragmatic els is extremely uncommon with an estimated incidence of 2 - 5% of pulmonary sequestration , and of 10 - 15% for els below the diaphragm ( 2 ) .
it is most frequently seen in the first 6 months of life , but is found incidentally , though rarely , in older children and adults ( 3 ) .
grossly , els is usually a single pyramidal to round or oval lesion , ranging from 0.5 - 15 cm in largest diameter .
it is covered by a smooth to finely wrinkled pleura , and may be adherent to adjacent structures if previously infected ( 3 ) .
the arterial supply in 80% of esl cases comes directly from the thoracic or abdominal aortas ; around 15% receive blood from another systemic artery ( celiac , renal artery ) and 5% from the pulmonary artery , and venous drainage is predominantly into the systemic circulation , though about 25% drain completely or partially via the pulmonary veins ( 4 ) . in most cases
the sequestration is close to the diaphragm in the left suprarenal area , and is associated with other congenital malformations in approximately 50% of cases .
diaphragmatic hernia , pericardial defects , pectus excavatum , and congenital heart disease are the most frequently reported associated anomalies ( 5 ) .
most els are asymptomatic . however , the lack of communication between an els and the tracheobronchial tree results in fluid accumulation in sequestered tissue , which results in a large size and extrinsic compression .
they rarely have a fistulous communication with the respiratory or gastrointestinal tract , and thus cystic overinflation and infection are unlikely . to our knowledge , this is the first report of infradiaphragmatic retroperitoneal els accompanied with infection , but we were unable to find any fistulous tract to the respiratory or gastrointestinal tract and any infection source .
spontaneous regression has been reported in patients with intralobar or extralobar intrathoracic sequestration ( 7 ) and several hypotheses have been suggested to explain spontaneous involution of the sequestered lung .
progressive fibrosis of dysplastic tissue , or thrombosis and fibrosis of the feeding vessel leading to progressive infarction of the abnormal lung would seem to be the most feasible ( 7 ) .
the treatment of infradiaphragmatic els is controversial , however , operative resection is still the mainstay of treatment .
removal allows confirmation of diagnosis and reassures that a neuroblastoma or a cystic adenomatoid malformation is not being left untreated .
resection also eliminates any risk of malignant transformation , which has been reported in other congenital cystic lung lesions ( 8) .
in addition , exploration allows the opportunity to address associated anomalies , such as , congenital diaphragmatic hernias . in summary , we describe the first case of an infected infradiaphragmatic retroperitoneal els , which occurred in a 32-yr - old man , and which presented with epigastric discomfort and fever .
we administered a staged management of the infected els and achieved an excellent clinical course . | infradiaphragmatic extralobar pulmonary sequestration is an extremely rare congenital malformation .
it is more frequently diagnosed in the antenatal period due to routine ultrasonic examination of the fetus or in the first 6 months of life , though on rare occasions it is discovered incidentally in adults .
a 32-yr - old man presenting with epigastric discomfort and fever was referred .
computed tomographic scanning showed that a 16-cm , multiseptated , dumbbell - shaped , huge cystic tumor was located beneath the diaphragm . on the next day
, 850 ml of thick yellowish pus was drained by sonography - guided fine needle aspiration for the purpose of infection control and diagnosis , but no microscopic organisms were found in repeated culture studies .
surgical removal of the cyst was performed through thoracoabdominal incision and most of these pathologic lesions were removed but we could not find the feeding arteries or any fistulous tract to surrounding structures .
histopathologic study revealed that it was extralobar pulmonary sequestration and culture study showed that many wbc and necrotic materials were found but there were no microorganisms in the cystic contents .
we report the first case of an infected infradiaphragmatic retroperitoneal extralobar sequestration which was administered a staged management and achieved an excellent clinical course . |
following their activation , memory b lymphocytes differentiate into plasmablasts , namely , a preplasma like - cell , which can simultaneously proliferate and secrete antibodies .
plasmablasts can quickly differentiate into short - lived plasma cells that secrete high amounts of antibodies , more than 10.000 molecules per cell per second [ 2 , 3 ] .
an increase in reactive oxygen species secondary to this important protein synthesis causes an oxidative stress responsible for their short lifespan [ 4 , 5 ] .
igg short - lived plasma cells are known to migrate towards infection sites , while iga populations favor mucosal tissues [ 6 , 7 ] . a germinal center reaction will generate memory b lymphocytes as well as a second wave of memory plasma cells , which will strengthen the initial wave of short - lived plasma cells that may migrate and persist in survival niches in the bone marrow . to reach the bone marrow microenvironment ,
plasma cells need to express the protein c - x - c chemokine receptor 4 ( cxcr4 ) [ 10 , 11 ] , a transmembrane activator , and caml interactor ( taci ) [ 12 , 13 ] .
these receptors bind c - x - c chemokine ligand 12 ( cxcl12 ) and a proliferative induced ligand ( april ) [ 11 , 14 ] .
furthermore , as plasma cell niches found in the bone marrow are limited in number , a competition occurs within resident cells and newly formed cells to replenish the long - lived plasma cell pool [ 15 , 16 ] .
bone marrow niches involved in plasma cell survival are highly complex environment characterized by a very low level of oxygen ( po2 ) , ranging from 1 to 6% , depending on blood vessel vicinity [ 17 , 18 ] .
these niches include several cell populations , such as megakaryocytes , basophils , monocytes , macrophages , dendritic cells , and t - cells .
mesenchymal stem cells ( msc ) as well as eosinophils are also present in bone marrow niches and appear to be essential for plasma cell survival [ 9 , 2527 ] .
eosinophils secrete il-6 as well as april , both promoting the maintenance and survival of plasma cells in the bone marrow for several years . in the bone marrow
, eosinophils are found to be highly proliferative and located in the vicinity of plasma cells [ 26 , 27 ] .
msc secrete cxcl12 [ 30 , 31 ] , which is involved in plasma cell migration as well as survival in bone marrow niches [ 32 , 33 ] .
msc secrete transforming growth factor beta-1 ( tgf-1 ) and tgf-2 , involved in b - cell homeostasis and death regulation in mouse models [ 34 , 35 ] .
in addition , msc can synthetize extracellular matrix molecules such as laminin , heparan sulfate , and elastin [ 22 , 29 , 36 ] . as reported in a mouse model study ,
the ability of plasma cells to adhere to their bone marrow niche is necessary for their survival and can be impaired by anti - vla-4 and anti - lfa treatments . besides , vcam-1 and icam-1 , which are natural ligands for vla-4 and lfa , respectively , are present on msc and are involved in cell - cell and extracellular matrix interactions .
the requirement for msc in plasma cell bone marrow niches is well - established [ 9 , 27 ] .
msc from human bone marrow were shown to inhibit proliferation [ 40 , 41 ] and b - lymphocyte ig secretion following polyclonal activation with cpg .
in contrast , msc have been shown to promote lps - activated b - lymphocyte igg secretion .
currently , studies on the generation of differentiated b lymphocytes are often based on in vitro interaction with cd154 .
these culture models support the differentiation of naive [ 4548 ] or activated b lymphocytes [ 4951 ] upon cd40 activation in the presence of a cocktail of cytokines such as il-4 , il-10 , and il-6 .
such in vitro generated plasma cells are often close to a short - lived phenotype , showing very high levels of ig secretion and a limited capacity to proliferate . in fact , the majority of in vitro models generate short - lived plasma cells .
actually , only a few studies have reported the successful generation of long - lived plasma cells in vitro [ 52 , 53 ] .
the goal of this study was to establish culture conditions enabling the differentiation of switched memory b cells into plasma cells using a bone marrow - like environment .
for this purpose we used a defined serum - free medium to avoid undesired interactions with animal proteins and to exert maximum control over the components present in the medium . here , we describe a three - phase culture system which generates high proportions of cd31cd38cd138 plasma cells , using interactions with human bone marrow mesenchymal stem cells .
this study has been approved by hma - qubec 's research ethics committee . regular platelet donors who agreed to participate in this study
have all signed an informed consent . peripheral blood mononuclear cells ( pbmc ) from healthy donors were recovered from leukoreduction chambers of plateletpheresis apparatus ( trima accel , terumo bct , lakewood , co ) and stored frozen as previously described . switched memory cd19 b lymphocytes were isolated from pbmc by a two - step negative selection using the easysep b - cell enrichment kit and easysep custom cocktail removing all igd- and igm - positive cells ( stemcell technologies , vancouver , canada ) , as previously described .
the purity of cd19igdigm intact switched memory b lymphocytes was higher than 95% , as determined by flow cytometry .
mesenchymal stem cells from human bone marrow were obtained from lonza ( walkerville , md , usa ) . human bone marrow mesenchymal stem cells ( msc ) were cultured in -mem from sigma - aldrich ( oakville , on , canada ) with 10% fetal bovine serum from life technology ( burlington , on , canada ) and 2 mm glutamax from thermo scientific ( rockford , il , usa ) .
a master bank was prepared by expanding the cells for 7 days using a quantum cell expansion system from terumo bct ( lakewood , co , usa ) .
the msc population was homogenuous , with > 95% of the cells of phenotype cd73cd90cd105 , as determined by flow cytometry .
these adherent cells were cultured in iscove 's modified dulbecco 's medium ( imdm ) supplemented with 5% low igg fetal bovine serum ( fbs ) , both from life technologies .
l4.5 cells were irradiated with 7500 rad using a gammacell 1000 elite cs--irradiator ( nordion international , kanata , canada ) to prevent their proliferation .
the human eosinophilic cell line eol-1 was obtained from dsmz ( acc 386 ; braunschweig , germany ) .
they were cultured in roswell park memorial institute ( rpmi ) medium with 10% low igg fbs , from life technologies . after 10 days of culture , eol-1 cells were activated for 9 days with 0.1 mm dibutyryl cyclic adenosine monophosphate ( dbcamp ) from sigma - aldrich . switched memory b lymphocytes were cultured in two different media .
the first one , termed fbs , was made of imdm , 10% ultra - low igg fetal bovine serum ( fbs ) , and 1% its ( 10 g / ml human insulin , 5.5 g / ml human transferrin , and 6.7 ng / ml sodium selenite ) , all from invitrogen ( burlington , on , canada ) . the second medium is a bovine protein - free medium ( bpfm ) .
in addition to the supplements added to imdm , bpfm was supplemented with 60 ng / ml of long - r3 insulin - like growth factor 1 ( igf-1 ) , 100 m of trolox , and 5.5 g / ml of human holotransferrin from sigma - aldrich ; lipid mixture 1 ( lp-1 ) , composed of 2 g / ml arachidonic acid , 10 g / ml of each of linoleic , linolenic , myristic , palmitic , stearic , and oleic acids , 220 g / ml cholesterol from new zealand sheep 's wool , 2.2 mg / ml tween-80 , 70 g / ml tocopherol acetate , and 100 mg / ml pluronic f-68 from sigma - aldrich ; 20 g / ml of low density lipoprotein ( ldl ) from stemcell technologies ; 250 g / ml of 5% human serum albumin ( hsa ) from bayer inc .
( toronto , on , canada ) ; and 10 g / ml of human recombinant insulin from life technologies .
cytokines were added following a three - step culture protocol . during the expansion phase , 50 u / ml of interleukin 2 ( il-2 ) , 120 u / ml of il-4 , and 20 u / ml of il-10 , all from peprotech ( rocky hill , nj , usa ) , were added . in these conditions ,
irradiated l4.5 cells were seeded with switched memory b cells at a ratio of one l4.5 to 5 b lymphocytes ( 1 : 5 ) to sustain proliferation for 8 to 12 days [ 45 , 59 ] . for the 4-day long second phase ,
called transition phase , 10 u / ml il-6 ( from peprotech ) was added to the medium to induce switched memory b - cell differentiation .
in addition , the l4.5 : b - cell ratio was reduced to 1 : 20 .
the last step , the differentiation phase , was performed in the presence of 10 u / ml il-6 and 20 u / ml il-10 .
control was performed with l4.5 cells at a constant 1 : 20 ratio . at the end of the culture ,
irradiated l4.5 cells represent less than 5% of the total number of harvested cells ( data not shown ) .
assays in the presence of activated eol-1 cells were achieved at a 1 : 20 ratio with switched memory b lymphocytes in a 6.5 mm polycarbonate transwell with 5 m pores ( fisher , pittsburgh , pa , usa ) .
eol-1 cells were seeded in the top well , while switched memory b lymphocytes were seeded in the bottom well in presence of l4.5 cells at a ratio of 1 : 20 .
msc were seeded with b lymphocytes at a 1 : 5 ratio . in transwell conditions
, msc were plated in the top well , with or without activated eol-1 . for direct contact assays
, msc were seeded in primaria 24 wells ( fisher ) 1 hour before the addition of b lymphocytes .
the differentiation phase was performed for 8 days next to the transition phase of cell culture .
all conditions tested were run in parallel at 37c with 5% co2 , under atmospheric o2 conditions ( 18% ) and normoxic physiologic conditions with 8% o2 using a heracell 150i incubator ( thermo scientific ) . for assays at 8% o2
, media were preincubated for 12 to 24 hours in the absence of cytokines before the start of the culture to reach gas pressure equilibrium . between each step , switched b lymphocytes were centrifuged at 200 g to remove the supernatant containing the previous cytokine cocktail . during the culture process
, media were changed every 2 days , and corresponding cell lineages were replaced every 4 days .
cell viability was measured on a nucleocounter nc-250 , after labeling with ao solution 18 following the manufacturer 's instructions ( chemometec , allerod , denmark ) .
igg and iga content were measured in the culture supernatant by standard elisa assay as previously described .
determination of the phenotype of b lymphocytes was performed every 4 to 8 days by flow cytometry during expansion , transition , and differentiation phases . on day 4
, cultured b lymphocytes were analysed to confirm their purity by targeting cd3 , cd14 , cd19 , cd45 , cd154 , iga , igd , igg , and igm markers .
viability was assessed following a 30-minute staining protocol using pacific blue succinimidyl ester ( pb ) .
cells were washed with pbs supplemented with 1% fbs and 0.001% nan3 and then stained with the appropriate antibodies listed above .
allophycocyanin- ( apc- ) conjugated anti - cd3 and anti - igg and phycoerythrin- ( pe- ) conjugated anti - igd and anti - cd154 were obtained from bd biosciences ( san jose , ca , usa ) .
fluorescein isothiocyanate- ( fitc- ) conjugated anti - cd14 , anti - igm , and pe - conjugated anti - cd45 were from ebiosciences ( san diego , ca , usa ) and fitc - conjugated anti - iga was from bio - rad ( mississauga , on , canada ) . to assess their differentiation status ,
cells were stained with antibodies targeting cd19 , cd31 , cd38 , cd39 , and cd138 markers .
fitc - conjugated anti - cd31 ( clone wm59 ) , apc - conjugated anti - cd39 , and efluor450-conjugated anti - cd38 ( clone hb7 ) were all from ebiosciences .
pe - conjugated anti - cd138 ( clone ba-38 ) was from abcam ( toronto , on , canada ) , pe - cy7-conjugated anti - cd105 ( clone 43a3 ) from biolegend ( san diego , ca , usa ) , and pe - cy7-conjugated cd19 from bd biosciences .
data analyses were performed directly after labeling with a partec cyflow ml flow cytometer ( swedesboro , nj , usa ) and flowmax 3.0 software ( partec , mnster , germany ) . for each experiment ,
dead cells were excluded from analysis , and at least 10 000 events were recorded .
analyses were performed with fcs express 4 software ( de novo , los angeles , ca , usa ) , and positivity for each marker was determined by fluorescence minus one ( fmo ) .
in addition , b - cell proliferation was measured with cellvue burgundy , following the manufacturer instructions ( ebioscences ) for a period of 4 days .
an initial measure performed on day 0 was used as a reference for the assay .
the oxidation - reduction potential in culture medium was measured with a micro - pt / agcl combination mi-800/410 cm redox electrode ( microelectrodes , inc . ,
bedford , nh , usa ) connected to a phi model 510 ph meter ( beckman coulter , mississauga , on , canada ) .
measures were performed directly after medium preparation , and analyses in culture supernatants were done at the end of the cultures .
cytokines and immunoglobulins were measured in culture supernatants by a multi - analyte profiling technology using luminex is 200 system ( luminex , austin , tx , usa ) .
each of the targeted molecules was measured separately on flat - bottom immulon - ii plates using bio - plex pro human , inflammation panel 1 april / tnfsf13 , sdf1 + , ip-10/cxcl10 , th17 , il-21 , tgf-1 , and tgf-2 ( all from bio - rad ) .
bio - plex pro reagent kit iii was used for april , cxcl12 , and cxcl10 , bio - plex pro reagent kit ii was used for il-21 , and bio - plex pro reagent kit i for tgf-. iga , ige , igm , and igg subclasses were measured using bio - plex pro human isotyping kit ( bio - rad ) .
all analyses were done following the manufacturer 's instructions . in order to visualize cellular heterogeneity from a single cell cytometry assay ,
data for plasma cells ( day 8) were run on the spade analysis software ( cytobank , inc , http://www.cytobank.org/ ) .
analyses were performed on viable cells , classified in 50 nodes , with each one gathering cells according to the similarities in their phenotype .
data provided represent the mean of global medians used for the standardization of results from each experiment , and the color scale was set to asymmetric .
all statistical analyses were done using graphpad instat version 3 ( graphpad software , san diego , ca , usa ) .
we have previously established that the presence of il-2 , il-4 , and il-10 allows long - term expansion of cd40-activated switched memory b lymphocytes in the presence of bovine serum .
besides , we reported that our in - house serum - free medium , namely , bovine protein - free medium ( bpfm ) , was able to sustain the proliferation of cd40-activated b lymphocytes , including naive and memory cells , using the same cytokine cocktail .
the next steps were done to evaluate the outcome of switched memory b lymphocytes when cultured in bpfm .
purified cd19igdigm cells were cultured in parallel for 12 days in bpfm and in a medium containing 10% fbs in the presence of cd154 l4.5 cells and il-2 , il-4 , and il-10 ( figure 1 ) .
the proliferation rate was calculated for 3- to 4-day periods to monitor the progression of cd19igdigm cells ( figure 1(a ) ) .
overall , during this three - step culture , the cells were expanding similarly in both conditions ( bonferroni multiple comparisons test , p > 0.05 ) , giving an average of 5.3 0.2-fold expansion in bpfm and 5.8 0.1-fold expansion in the presence of fbs .
total expansion , starting from 1 10 seeded cells , had reached 82- to 429-fold for cells cultured in fbs and 71-to 328-fold in bpfm ( data not shown ) .
the presence of fbs was slightly advantageous for the switched activated b lymphocytes in regards to total expansion ( paired t - test , p = 0.0118 ) . viability assessment did not show any significant differences when comparing both conditions ( figure 1(b ) ) ( dunn 's multiple comparison test ; p > 0.05 ) , decreasing on day 12 to 77 2% and 72 2% in fbs and bpfm , respectively .
the cells were maintained in culture for an additional 9 days to measure their commitment towards differentiation by measuring the secretion of igg and iga ( figure 1(c ) ) .
iga secretion was similar in both conditions , reaching 14.4 4.9 g / ml and 15.2 5.6 g / ml , in fbs and bpfm , respectively .
igg secretion appeared higher when cells were cultured in the presence of fbs ( 122.5 28.8 g / ml ) than in bpfm ( 72.3 17.2 g / ml ) ; however this difference was not statistically significant ( dunn 's multiple comparisons test , p > 0.05 ) .
the progression towards differentiation was also monitored on day 12 according to cd31 , cd38 , cd39 , and cd138 expression ( figure 1(e ) ) .
overall , the cellular phenotype was similar in both conditions , except for the proportion of cd38 cells , which was lower in cells cultured in bpfm ( 39% 8% , compared to 75% 8% in fbs ) ( unpaired student 's t - test , p 0.05 ) .
finally , the measure of redox potential in both media and in cell culture supernatants showed no significant differences ( figure 1(d ) ) .
overall , we showed that bpfm allows switched memory b lymphocytes to proliferate and to initiate differentiation .
this medium was thus used to further investigate the in vitro generation of plasma cells .
noticeably , the significant decrease in the proportion of cd38 cells had no impact on the smaller cd38cd138 cell population .
b lymphocytes were pushed into differentiation in bpfm using a simple three - step model involving a shift in the l4.5 : b - cell ratio and modifications of cytokines , as previously described ( figure 2(a ) ) . as previously observed , cd38 and cd39 expression rapidly increased following b - cell activation ( figure 2(b ) , d8 ) . however , cd38 expression decreased during the transition and differentiation steps .
this decrease was related to the absence of retinoic acid in the bpfm medium ( data not shown ) , as already reported for cd34 cells .
besides , transition towards differentiation resulted in a slight increase in the number of cells expressing the cd31 and cd138 markers ( figure 2(b ) ) . at the end of the differentiation phase ,
most of the cells were still positive for cd39 ( > 85% ) and about half of them were also positive for cd31 , cd38 , and cd138 .
overall , subjecting cells to an 8% o2 level resulted in phenotypes similar to what is obtained with the standard 21% o2 condition .
the igg content appeared higher at 21% o2 ( 73 8 g / ml ) than at 8% o2 ( 25 6 g / ml ) .
iga content was similar for both conditions , namely , 10 7 g / ml and 12 7 g / ml , respectively , for 21% and 8% o2 ( data not shown ) .
finally , monitoring of cell division using cellvue staining showed evidence for a significant decrease , by about 2- to 3-fold , in the proportion of dividing b lymphocytes during differentiation at 21% and 8% o2 ( figures 2(c ) and s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7801781 ) . in this assay ,
cell viability varied from 80 1.5% and 75 1.7% for 21% and 8% o2 , respectively ( data not shown ) .
overall , the above results confirm that the bpfm medium is allowing efficient differentiation of b lymphocytes when combined with a low cd154 interaction level in the presence of il-6 and il-10 .
the purpose of the next experiment was to model the environment found in the bone marrow , using an activated eosinophil cell line , namely , eol-1 , and human bone marrow mesenchymal stem cells , during the differentiation phase ( figure 3 ) .
for these assays , b lymphocytes were maintained in direct contact with l4.5 cells ( l ) , while eol-1 ( e ) and/or msc ( m ) were added in the upper chambers of transwell plates .
the proportions of cd38cd138 cells , cd38cd138 cells , and cd31 cells within these subpopulations of plasma cells were determined in viable cells at the end of the differentiation phase , on day 20 ( figure 3(a ) ) .
no significant differences were observed in total cell numbers within cultures done at 8% o2 and 21% o2 for all combinations tested .
in contrast , the proportions of cd38cd138 cells and cd38cd138 cells were greatly influenced by the composition of the coculture partners ( figures 3(b)-3(c ) ) .
differentiating cells subjected to 21% o2 showed that cd154 activation alone or in combination with eol-1 cells allowed the formation of , respectively , 13.9 3.5% and 14.8 2.3% of cd38cd138 cells .
in this condition , the addition of msc to cd154 and eol-1 cells ( lem ) significantly increased the proportion of cd38cd138 to 27.1% 4.4% ( dunn comparison t - test with p 0.01 ) .
when cells were subjected to 8% o2 , no differences were observed between the addition of eol-1 to l4.5 cells and l4.5 cells alone ( 9.6 0.6% and 12.4 2.5% ) , while a significant increase was observed when msc were added ( dunn comparison t - test with p 0.05 ) .
furthermore , cd154 activation alone allowed the generation of 3,4 0,8% cd38cd138 cells , which was significantly less than activation with lem ( 11.6 4,3% , p 0.05 ) ( figure 3(c ) ) .
the presence of eol-1 cells and msc also resulted in a significant enhancement of differentiation in regards to the proportion of cd31 cells for cultures done at either 21% or 8% o2 within the cd38cd138 cell population .
furthermore , the effect was more striking at 21% o2 , since the proportion of cd31 cells increased to 55.2 10.4% with eol-1 alone and to 85.1 1.4% when both cells were added to the upper wells ( p 0.05 ) ( figure 3(d ) ) . in contrast , the expression level of cd31 within the cd38cd138 cells was systematically higher than 75% in all conditions tested ( figure 3(e ) ) .
finally , the total populations of cd138 and cd138 cells were favored when msc were added to eol-1 and l4.5 cells , increasing by almost 2-fold at 49.4 4.7% ( 21% o2 ) and 23.7 2.6% ( 8% o2 ) , respectively , when compared to l4.5 cells alone ( 23.2 5.2% ( 21% o2 ) and 13.1 2.9% ( 8% o2 ) , figures 3(f ) and 3(g ) ) .
overall , these results suggest that msc appear to enhance differentiation of switched memory b lymphocytes .
msc appear to be able to secrete factors which synergize with cd154 stimulation to increase the proportion of plasma cells .
since the addition of msc to switched memory b - cell cultures seems to promote the emergence of plasma cells , we have measured the influence of msc using direct contact and transwells in the presence or absence of l4.5 cells ( figure 4 ) .
the outcome of the cells was monitored as above , using 8% and 21% o2 levels .
the proportions of cd38cd138 cells ( figures 4(a ) and 4(b ) ) and cd31cd39 cells ( figures 4(c ) and 4(d ) ) were evaluated for all conditions .
for the four coculture conditions tested , no differences were observed between the two oxygen levels .
the addition of msc in direct contact or in indirect contact using a transwell plate was the most potent stimulus for the emergence of cd38cd138 cells .
a direct interaction of switched memory b lymphocytes with msc allowed the differentiating cells to reach about 50% of the cultured cells , which was significantly higher than with l4.5 cells alone ( figure 4(a ) ) . furthermore , when msc were added in the upper chambers of transwells , the proportions of cd38cd138 were comparable to those obtained when in direct contact , reaching 40.2% 8.2% and 45.7% 7.9% in 8% and 21% o2 , respectively . however , the expression of cd138 was much higher , according to mfi , when msc were in direct contact when compared to the transwell setting ( figure 4(f ) ) .
overall , an increase of at least 20-fold of cd138 mfi was observed in 5 independent experiments . besides , when l4.5 cells were present in the lower chambers , we noticed a significant decrease , by about 2-fold , in the proportions of cd38cd138 cells ( figures 4(a ) , 4(b ) , and 4(e ) ) , suggesting an inhibitory effect of l4.5 cells . on the other hand ,
the proportion of cd31cd39 cells reached at least 80% and significantly increased in all conditions whenever msc were present , with or without direct contact with b lymphocytes ( figures 4(c)-4(d ) ) .
overall , a 2-fold increase was noticed compared to cocultures done with l4.5 cells alone , which yielded about 40% cd31cd39 cells at either 8% or 21% oxygen . for this cellular phenotype , no negative effect of l4.5 cells , present in the lower chamber ,
cocultures of switched memory b lymphocytes were also done with activated eol-1 cells alone and compared to l4.5 cells alone , as well as the above conditions with msc .
our results from two independent samples showed that eol-1 cells were unable to maintain the viability of b lymphocytes .
viability was lower than 40% 1.2% and decreased rapidly from day 12 to day 20 .
nevertheless , we also noticed that the proportion of residual cells being able to differentiate into cd38cd138 was much lower than that observed with l4.5 and msc ( data not shown ) .
these observations indicate that msc alone efficiently promotes the differentiation of switched memory b cells into plasma cell , independently of oxygen concentration .
our results also indicate that msc could secrete soluble factors contributing to this differentiation - promoting effect .
finally , direct contact with msc generated plasma cells showing a higher level of cd138 expression .
the above results established that msc stimulation enhances plasma cell generation under either 8% or 21% o2 levels .
furthermore , analyses of the cell profiles according to the fsc and ssc parameters also revealed two distinct populations ( figure 3(a ) ) .
we thus further characterized these subsets by delineating the fsc ssc as population a and fcsssc as population b , as indicated by the respective gates shown in figures 5(a ) and 5(b ) .
all analyses were done at the end of the differentiation phase , and cells were cultured in direct contact with msc ( figures 5(b ) , 5(d ) , and 5(f ) ) or l4.5 cells ( figures 5(a ) , 5(c ) , and 5(e ) ) and subjected to both oxygen conditions .
overall , about 50% of all cells analysed were present in population b ( fcsssc ) when the cells were cultured with msc .
however , the frequency of fcsssc was significantly lower in the presence of l4.5 cells , reaching about 10% and 15% in 8% and 21% o2 , respectively ( dunn 's multiple comparisons test ; p < 0.05 ; data not shown ) .
additionally , we have observed important variations in regards to the expression of cd31 , cd38 , cd39 , and cd138 within the a and b populations .
these variations were independent of the coculture conditions , namely , l4.5 cells or msc , and oxygen levels ( figure 5 and table 1 ) .
overall , population a cells showed low expression of cd31 , cd38 , and cd138 ( figures 5(c)5(f ) ) and low frequencies of cells positive for these markers ( table 1 ) .
in contrast , cells forming population b were characterized by a high expression of cd31 , cd38 , and cd138 , which are hallmarks of plasma cells .
furthermore , cells found in gate b included the highest proportion of cd138 cells ( 75% ) and cd38cd138 cells ( 85 1.2% , data not shown ) when cultured in the presence of msc ( table 1 ) . to highlight this shift from a to b
, we then compared the frequency of cd38cd138 populations inside each gated subset compared to their content in the total ( a + b ) population ( figure 5(g ) ) .
the proportions of fscssccd38cd138 cells ( b population ) reached 38.4% 1.9% and 30.1% 3.2% of total cd38cd138 cells , following culture in the presence of msc at 21% and 8% o2 , respectively .
culturing cells in the presence of l4.5 cells led to lower proportions of plasma cells , as only 10.9% 3.9% and 13.7% 2.4% of cd38cd138 cells were observed at 21% and 8% o2 , respectively ( p 0.001 in both conditions ) .
moreover , a comparison of viabilities in regards to a and b populations ( figure 5(h ) ) showed that the fscssc cells were characterized by the highest viability when cultured with msc or l4.5 cells .
however , low viability was observed for cells in population a when cultured with msc , with 35.6% 9.6% and 23.7% 53% viable cells for 21% and 8% o2 , respectively .
in contrast , cells in populations a were still viable at 70% when cultured with l4.5 cells .
we also observed that the cd138 cells generated at the end of the differentiation phase were characterized by low expression of cd19 and were also predominantly positive for cd49d ( vla-4 ) , which is very important for interactions with the bone marrow cellular matrix ( data not shown ) .
finally , intracellular levels of expression of / light chains were verified in three independent experiments for cd138 and cd138 cells corresponding to a and b populations ( data not shown ) .
we observed that all cd138 cells express intracellular and light chains , in proportions of 40% and 60% , respectively .
however , only 70% of cd138 cells were positive for either or light chain expression , suggesting heterogeneity in the a but not in the b population . once again
, these results emphasize the ability of bone marrow msc to favor plasma cell generation in bpfm medium , but they do not show any impact related to o2 levels .
noticeably , the distinct population characterized by fscssc includes a majority of plasma cells with a high viability which are better supported by the presence of msc .
the above results highlight that the differentiation phase with msc and l4.5 cells generates two distinct populations of plasma cells .
thus , the heterogeneity of the a and b populations was further investigated using spade analysis .
this tool enables us to directly visualize the relative expression of cd31 , cd38 , cd39 , and cd138 ( figures 6 and s2 ) .
spade 2-dimensional representation underlines the distinction of the a and b populations inside gated viable cells .
the nodes representing population b , namely , fcsssc , are showing higher expression of cd31 , cd38 , and cd138 when compared to the nodes of population a ( figure 6 ) .
we observed , similarly to what was shown above , that msc were more efficient in maintaining this plasma cell population than l4.5 cells ; yet no distinctions were seen according to oxygen levels .
the observations ( figures 3 and 4 ) that switched b lymphocytes were directed to differentiate towards plasma cells when cultured in the presence of msc , even without direct contact , suggested the involvement of soluble factors in the differentiation - promoting effect .
thus , further evaluations of culture supernatants using multiplex luminex assays were performed to measure their contents in il-21 , cxcl10 , cxcl12 , tgf-1 , tgf-2 , and april at the end of the differentiation phase after coculture with l4.5 cells or with msc , in either direct contact or transwell ( figures 7(a ) and 7(b ) ) .
our results revealed that il-21 , cxcl10 , tgf-2 , and april were in negligible amounts for all tested conditions ( data not shown ) .
in fact , we were able to detect only cxcl12 and tgf-1 in culture supernatants , and the same secretion patterns were observed for both o2 levels .
the quantities of cxcl12 were significantly higher when msc were in direct contact with the differentiating cells .
curiously , supernatants of msc added in transwell without contact did not contain comparable amounts of cxcl12 than direct cocultures .
we determined that msc , when cultured alone ( ctrl ) , can secrete 276 129 ng / ml and 335 112 ng / ml of cxcl12 at 21% and 8% o2 , respectively .
noticeably , when b lymphocytes were cultured in 21% o2 in contact with msc , the secretion of cxcl12 reached 315 119 ng / ml ; this concentration that was reduced by about 50% ( 148 32 ng / ml ) was detected when the culture was done in 8% o2 .
we observed very high variations among samples for the amount of tgf-1 ( figure 7(b ) ) .
however , tgf-1 secretion appeared significantly different only when secretion of msc alone was compared to that of b lymphocytes cultured in the presence of either l4.5 cells or msc in transwell assays done at 8% or 21% o2 .
furthermore , conditions leading to reduced levels of cxcl12 and tgf-1 , for example , in transwell assays or in 8% o2 , may suggest a variation in the b lymphocytes ' capacity to use soluble factors .
the functional characteristics of the differentiated cells were further characterized by evaluating their secretion of immunoglobulins ( figures 7(c)7(g ) ) . as expected , igg1 was the highest and the main isotype detected in our supernatants for all conditions tested .
igg4 and iga were detected in all conditions , with no differences between coculture conditions or oxygen levels .
the most significant difference , an almost 10-fold increase , was observed for igg1 secretion by cells cultured in direct contact with msc compared to those cultured with l4.5 cells , regardless of the oxygen level ( figure 7(c ) ) .
however , at 21% oxygen , igg1 secretion was significantly higher when cells were in contact with msc ( 66.5 12.9 g / ml ) than in transwells ( 31.1 7.2 g / ml ) .
igg2 and igg3 secretion levels were higher when cells were in contact with msc compared to l4.5 cells in 21% o2 ( figures 7(d ) and 7(e ) ) . furthermore , we have estimated the secretion rate according to total igg content using the seeding density on day 16 . at that point , cells were washed and put back in fresh culture medium for four supplemental days .
the estimated secretion rate is 18 pg of igg per cell per day ( data not shown ) .
collectively , these data confirm that msc are very efficient at supporting the generation of functional ig - secreting cells displaying several characteristics of fully differentiated plasma cells .
in this study , we show that msc allow the differentiation of human switched memory b cells into cd38cd138 plasma cells in a serum - free medium . to our knowledge , this is the first model allowing the generation of cd31cd38cd138 cells in the presence of msc from human bone marrow and the first report of such differentiation in the absence of bovine serum . in addition , the newly generated plasma cells are mainly nondividing cells and gain ig - secreting function .
the detection of cxcl12 and tgf-1 secreted by msc validates the usefulness of the in vitro model aiming to reproduce an environment similar to plasma cell niches found in the bone marrow .
our results also highlight the differential effects of cd40 or msc stimulation on the differentiation of human switched memory b lymphocytes . while msc favored the commitment towards cd31cd38cd138 plasma cells , l4.5 cells seem to be slowing down this process and to preferentially activate cells with an intermediate cd31cd38cd138 phenotype .
finally , we observed that the generation of newly formed plasma cells appears independent of oxygen levels in our model .
the in vitro generation of human long - lived plasma cells has been reported in two independent studies [ 52 , 53 ] .
cocco and collaborators were the first to report the long - term maintenance of plasma cells using a three - step culture allowing maintaining cells for up to 62 days [ 52 , 53 ] .
as a first step , they used polyclonal activation of peripheral blood cd27 b cells to generate plasmablasts .
the last step was designed to reproduce stromal environment using the gamma - irradiated fibroblastic mouse cell line m2 - 10b4 supplemented with il-6 and il-2 .
their model generated cd38cd138 cells which were characterized by expression of genes associated with long - lived plasma cells and were secreting igg at about 150 pg / cell / day .
the authors however reported that a rapid decrease in total viable cell numbers was observed during this last step , suggesting that persistent viable cells were not abundant in their model .
a second group also used purified peripheral blood cd27 cells , but in a four - step culture system .
, cd27 cells were first activated through soluble cd40 interaction in the presence of a polyclonal activator ( cpg ) and a mixture of il-2 , il-10 , and il-15 .
jourdan 's group was able to generate long - lived plasma cells using stromal cells purified from human tonsils , in the presence of il-6 and april .
long - lived plasma cells were generated by purifying cd138 cells obtained at the end of the third step and incubating these cells with stromal cells , with or without contact , for the last culture step . at the end of their 4-step process , jourdan and collaborators obtained newly formed cd38cd138 cells typical of long - lived plasma cells according to their gene expression pattern .
these authors also observed a rapid decrease in cell numbers during the final step of their culture system .
one unique study reports the differentiation of switched memory b lymphocytes using contact with bone marrow msc . in their study ,
traggiai and collaborators have cultured purified cells with msc , combined with stimulation by soluble cd40l and cpg .
interestingly , they found that msc favored igg secretion when b lymphocytes were in direct contact compared to a barrier system modeled by transwell assays .
noticeably , all of the above studies were performed with media containing bovine serum and used soluble cd40 and polyclonal activation in a separate first step or simultaneously with other cellular effectors [ 52 , 53 , 63 ] . in our model ,
the source of b lymphocytes is also peripheral blood , but the selection method we use includes cd27 igg- and iga - positive cells and excludes cd27igdigm cells , which are , respectively , absent and present in jourdan and cocco culture systems .
our culture model was initiated by an 8-day expansion step generating cd40-activated memory b lymphocytes [ 47 , 65 ] .
the expansion rate we obtained was at least 20-fold ; that is , starting with 1 10 switched memory b lymphocytes on day 0 , we could generate up to 20 10 cells . despite the fact that cells stopped to proliferate and viability decreased during the differentiation phase
, we were able to recover at least 5 10 cd138 cells on day 20 .
the last step of our culture system was based upon direct contact with viable human bone marrow msc in the presence of il-6 and il-10 .
we did not maintain the cells for longer periods , as we deliberately stopped cultures on day 20 , but the observed phenotype was similar to that reported by cocco and jourdan studies .
msc could favor plasma cell formation and survival by secretion of cxcl12 and tgf-1 and direct contact as reported in studies of the bone marrow microenvironment of plasma cells .
msc secretion was probably supported by the presence of il-6 in our culture medium and could have been increased under low oxygen conditions [ 6870 ] .
tgf-1 can favor the quiescent state of hematopoietic stem cells in the bone marrow and thus might also help to maintain the survival of plasma cells .
in addition , the newly generated plasma cells were almost all positive for cd31 , an adhesion molecule , supporting their commitment to respond to direct cellular interactions .
however , we did not add april in our culture medium and did not detect april in our coculture supernatants using l4.5 cells , eol-1 cells , or msc . such discrepancy with other studies
could be linked to the fine composition of the switched memory b lymphocytes used in our study , which included both cd27 and cd27 cells and excluded igd and igm cells . in our model , the newly generated plasma cells were secreting approximately 18 pg of igg per cell per day , which is quite similar to the rate reported by jourdan et al . for long - lived plasma cells . from a molecular perspective
, we can estimate that one cell is able to release an average of 800 igg molecules per second .
the secretion rate of short - lived plasma cells can reach up to 10 000 igg / sec / cell .
therefore , our observation of a low secretion rate is consistent with a long - lived character for our in vitro generated plasma cells .
furthermore , the absence of benefit for cultivating in 8% o2 could be explained by a very low exposure to oxidative stress , which is usually associated with a very high secretion of ig .
moreover , the direct contact with msc favored a low igg secretion and correlates with previous observations done with msc from bone marrow and b lymphocytes [ 63 , 72 ] .
further analyses revealed the emergence of two distinct populations , fcsssc and fscssc , from switched memory b lymphocytes .
these observations could be related to the hypothesis of a predefined fate upon differentiation related to asymmetrical divisions in germinal center ( gc ) reactions , leading to different fates for daughter cells .
such variability in fsc and ssc patterns has already been reported for plasma cell populations in regards to their cytoplasmic composition .
furthermore , we observed that the fcsssc cells were mainly cd31cd38cd138 , a phenotype that resembles early plasma cells .
although we have some indications that their secretion capacity could be different from that of fscssc cells , this has to be confirmed with sorted a and b cell populations using elispot or elisa methods . in conclusion , our study presents a culture model enabling the generation of plasma cells from switched memory b lymphocytes .
this model allows the production of large quantities of plasma cells by combining a short expansion step ( < 14 days ) and a differentiation phase in a bone marrow - like environment .
all cultures were done in the absence of bovine serum which , in the perspective of cellular therapy development , is very important for preventing adverse immunogenicity [ 7779 ] .
we believe that this model opens new avenues for autologous cell therapy for immunocompromised patients following cancer treatment or stem cell transplantation .
such plasma cells generated in vitro from activated switched memory b lymphocytes could attenuate the risk of infections in patients .
clinical studies have showed that 85% of bone marrow transplant patients will have at least one infection during the year that follows the graft , with a higher risk during the first 100 days .
furthermore , in our model , the newly formed plasma cells are exposed to human physiological oxygen levels [ 8284 ] , so they could adapt more rapidly and migrate more easily to bone marrow niches . however , additional investigations are needed to confirm the long - lived character of our in vitro generated plasma cells .
first , we plan to produce enough cells to detect blimp1 , irf4 , and xbp-1 proteins , as has been reported elsewhere [ 52 , 53 , 76 ] .
we are also currently investigating whether plasma cells generated in this model will be able to migrate to the bone marrow and reconstitute the secretion of human immunoglobulins in a nod - scid il2r ( null ) mouse model that is primarily designed for in vitro generated stem cell transplantation . | the differentiation of human b lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity .
however , the terminal differentiation and survival of plasma cells are still topics with much to be discovered , especially when targeting switched memory b lymphocytes .
plasma cells can migrate to the bone marrow in response to a cxcl12 gradient and survive for several years while secreting antibodies . in this study
, we aimed to get closer to niches favoring plasma cell survival .
we tested low oxygen concentrations and coculture with mesenchymal stem cells ( msc ) from human bone marrow . besides
, all cultures were performed using an animal protein - free medium .
overall , our model enables the generation of high proportions of cd38+cd138+cd31 + plasma cells ( 50% ) when cd40-activated switched memory b lymphocytes were cultured in direct contact with mesenchymal stem cells . in these cultures , the secretion of cxcl12 and tgf- , usually found in the bone marrow ,
was linked to the presence of msc .
the level of oxygen appeared less impactful than the contact with msc .
this study shows for the first time that expanded switched memory b lymphocytes can be differentiated into plasma cells using exclusively a serum - free medium . |
papillary thyroid carcinoma being the most common type of differentiated thyroid malignancy has a predilection for spread through the lymphatic system and thus commonly involves the central and lateral compartmental lymph nodes of the cervical region .
soft tissue metastasis is even rarer and amenable to radioiodine treatment similar to metastatic lesions in the lung and bones when they concentrate iodine .
the concerned case is unique in the sense that the involvement simultaneously of three soft tissue organs is present with different iodine - concentrating abilities , whereas a review of the literature did not find the simultaneous presence of iodine - concentrating metastasis in one organ , iodine - nonconcentrating metastasis in another organ , and multiorgan soft tissue metastasis in the same patient .
the case report also emphasizes possible hematogenous route of metastasis which is rare in papillary thyroid malignancy .
a 42-year - old male patient with a history of total thyroidectomy and bilateral cervical nodal dissection positive for papillary carcinoma thyroid was referred to our department for a follow positron emission tomography - computed tomography ( pet - ct ) scan for evaluation of lung nodules and muscle lesions that he developed five years after primary treatment . during the follow - up , prompted by elevated thyroglobulin levels , an iodine-131 whole body scan and single photon emission tomography - computed tomography ( spect - ct ) [ figure 1 ] showed positive lesions , one in the liver and another in the left gluteal region .
a fludeoxyglucose ( fdg ) pet - ct scan [ figure 2 ] was done to find more lesions and revealed metabolically active lesions in liver segment vi [ figure 3 ] and the left gluteus and a new paraspinal muscle lesion at the nape of the neck [ figure 4 ] , whereas the lung nodules were metabolically inactive [ figure 5 ] .
a post - therapy scan [ figure 6 ] at a therapeutic dose of 200 mci of iodine-131 revealed iodine concentration in the liver and a gluteal lesion , whereas there was no concentration in lung nodules and neck lesion .
the present fdg pet ct scan [ figure 7 ] as a response evaluation six month post treatment revealed a metabolic response in the liver and gluteal lesion [ figure 8 ] , but an increasing size of the neck lesion [ figure 9 ] and lung nodules [ figure 10 ] .
presently , the patient is put on redifferentiation therapy with sorafenib and suppressive doses of thyroxin .
the single photon emission tomography - computed tomography ( spect ct ) scan shows iodine - concentrating lesion in left gluteal muscle left gluteal lesions were metabolically active in pretherapy positron emission tomography - computed tomography ( pet ct ) study the new lesion at nape of neck fludeoxyglucose ( fdg ) activity but not lung nodules the 200 mci post - therapy scan concentration in liver and gluteal lesion but no concentration in neck lesion and lung nodules .
the positron emission tomography - computed tomography ( pet ct ) scan after six months iodine therapy shows treatment response in liver soft tissue deposit in the neck demonstrating increase in size and fdg uptake ( disease progression )
papillary thyroid carcinoma is the most common type of differentiated thyroid carcinoma accounting for at least 70% of all follicular cell - derived thyroid malignancies and is considered to be a relatively indolent tumor in which distant metastasis and death are rare .
the five - year survival rate for papillary thyroid cancer according to the stage is 100% for stage i and ii , 93% for stage iii , and 51% for stage
distant metastasis is the principal cause of death in cases of well - differentiated thyroid carcinomas .
about 10% of papillary carcinomas develop distant metastasis , with about 50% of patients having such metastasis at the time of diagnosis .
the prognosis of these patients is poor , and over 50% of the patients are likely to die within five years , irrespective of the histology of the tumor .
distant metastasis , although relatively uncommon , has been known to occur most commonly in the lungs , followed in frequency by bone and brain ( 0.1 to 5.0% ) .
isolated cases of soft tissue metastasis have been documented in the orbit , skull , skin , muscles , liver , spleen , pancreas , and adrenal gland.[613 ] metastatic lesions have also been documented in silent or occult thyroid neoplasms .
radioactive iodine treatment is considered to be the first line of treatment for distant metastasis from thyroid carcinomas that concentrate a significant amount of radioiodine . unlike our patient who developed metastatic disease and related symptoms
five years after treatment of primary disease , for patients who develop distant metastatic disease at the time of initial diagnosis , a positive iodine-131 whole body scan post treatment of primary lesions in the neck makes way for successful radioiodine treatment of metastasis . however , solitary metastatic lesions which do not concentrate radioiodine can be dealt with surgical removal and/or external beam radiation . to add to the confusion , our patient showed radioiodine concentration in one of the muscle lesions and liver but no concentration in another muscle lesion and lung nodules .
consequently , the muscle lesions responded to radioiodine treatment , whereas the neck lesion and lung nodules showed progression over the period of treatment .
the patient has been put on a suppressive dose of thyroxin and redifferentiation therapy . in the past ,
retinoic acid , thalidomide , and rosiglitazone showed efficacy in redifferentiation therapy of iodine - nonconcentrating thyroid malignancy metastatic lesions ; however , currently , sorafenib ( 400 mg twice daily ) and sunitinib ( 50 mg daily for 28 days followed by 14 days of no treatment per cycle ) , approved for other indications , show promise for thyroid cancer . | distant soft tissue metastasis and the simultaneous presence of iodine concentrating and nonconcentrating lesions in papillary thyroid cancer are extremely rare . the concerned patient , a histopathologically proven case of papillary thyroid cancer with nodal metastases treated with total thyroidectomy , bilateral cervical nodal dissection , and radioablation , subsequently developed lung , muscle , and liver metastasis .
triggered by increased thyroglobulin , the iodine-131 whole body scan and 200 mci iodine-131 post - therapy scan showed a left gluteus maximus lesion and a liver lesion .
fludeoxyglucose ( fdg ) positron emission tomography - computed tomography ( pet - ct ) scan intended to find additional lesions revealed iodine and fdg nonconcentrating bilateral pulmonary nodules and a single fdg avid hepatic and two muscle metastases .
although fdg concentration in metastatic pulmonary nodules is generally low , the ct characteristics were classical for metastatic lesion . a follow - up fdg pet - ct study six months after 200 mci iodine-131 radioablation showed treatment response in muscle and liver lesions but not lungs . |
oxanorbornadiene
dicarboxylate ( ond ) reagents are potent michael
acceptors , the adducts of which undergo fragmentation by retro - diels alder
reaction at rates that vary with the substitution pattern on the ond
moiety .
rapid conjugate addition between thiol - terminated tetravalent
peg and multivalent onds yielded self - supporting hydrogels within
1 min at physiological temperature and ph .
erosion of representative
hydrogel formulations occurred with predictable and ph - independent
rates on the order of minutes to weeks .
these materials could be made
non - degradable by epoxidation of the ond linkers without slowing gelation .
hydrogels prepared with ond linkers of equal valence had comparable
physical properties , as determined by equilibrium swelling behavior ,
indicating similar internal network structure . diffusion and release
of entrained cargo varied with both the rate of degradation of peg - ond
hydrogels and the hydrodynamic radius of the entrained species .
these
results highlight the utility of ond linkers in the preparation of
degradable network materials with potential applications in sustained
release . |
|
the interaction of these components , as well as the components themselves , are not fully understood .
this paper will discuss the three main regulatory paradigms involved in the regulation of cerebral blood flow : cerebral autoregulation , flow - metabolism coupling , and neurogenic regulation .
in addition , there are two cell types that have repeatedly been shown to play a central role in the regulation of cerebral blood flow : endothelial cells and astrocytes .
the process whereby the cerebral arteries ( specifically arterioles ) maintain a constant blood flow ( cbf ) in the face of changing cerebral perfusion pressure ( cpp ) is referred to as cerebral pressure autoregulation .
as shown below in figure 1 , between cpp pressures of 50150 mm hg cbf is relatively constant ; above and below these values , however , cbf varies markedly with cpp .
we view this phenomenon as independent of metabolic factors , thus this section is limited to changes in vascular tone as a result of cpp only .
flow - metabolism coupling is a distinct phenomenon that will be discussed in the following section .
several theories have been advanced , focusing on the endothelium , nerves , and the vascular smooth muscle itself .
because of the extensive investigation into the perivascular nerve fibers , discussion of this is relegated to a separate section below .
in addition , it has been proposed that the endothelium has mechanoreceptor properties that allow it to contribute to cerebral autoregulation .
the two main mechanical mechanisms that have been evaluated are shear stress and transmural pressure . increased flow - velocity ( shear stress )
. a similar endothelium - dependent response to increases in transmural pressure has also been demonstrated .
harder reproduced this work and also found that arterial constriction was associated with smooth muscle depolarization .
lastly , rubanyi showed that perfusate isolated from arteries that had been subjected to increased transmural pressure was capable of inducing vasoconstriction in nave vessels , implying some endothelial - derived factor .
stretch responses have also been theorized to originate in smooth muscle cells . originally formulated by bayliss in 1902 ,
the so - called myogenic hypothesis of cerebral autoregulation focuses on the mechanoreceptor properties of smooth muscle cells themselves .
the development in 1981 of isolated vessel techniques allowed the mechanisms to be separated from flow , neural , metabolic , and endothelial influences [ 5 , 6 ] .
recent work has focused on the transduction mechanisms between myogenic stretch and subsequent vasoconstriction , particularly the role of stretch - activated ion channels . since the first recordings of mechanosensitive ion channels in 1988 , a number of investigators have found evidence for these channels in vascular smooth muscle in a variety of tissues .
the electrical properties of these channels strongly suggest that they are nonselective cation channels [ 8 , 9 ] . the resultant membrane depolarization results in influx of ca through voltage - gated ca channels and smooth muscle constriction , a response that is abolished in the presence of inhibitors to voltage - gated ca channels .
it has also recently been shown that not only the rhoa - rho kinase pathway plays a pivotal role in cerebral artery mechanotransduction , but also the pathway is more active at progressively higher levels of stretch .
gokina and colleagues evaluated the effect of rho kinase inhibition on pressure autoregulation in cerebral arteries in the rat .
they found that administration of a specific inhibitor of rho kinase ( y-27632 ) selectively inhibited pressure - induced rise in intracellular ca as well as the development of myogenic tone .
studies have even demonstrated that calcium - independent mechanisms , so - called calcium sensitization , may be present as well .
this occurs when agonists lead to muscle contraction without a corresponding rise in intracellular calcium .
for over a century it has been appreciated that cerebral blood flow varies with cerebral metabolism .
this has most recently been shown with several functional imaging modalities , such as pet scanning and bold fmri .
so - called flow - metabolism coupling or functional hyperemia is perhaps the most clinically relevant of the cbf regulation paradigms , as cerebral tissue is among the least tolerant of ischemia .
interestingly enough , it has been shown that the generation of action potentials alone is not necessarily the main stimulus in flow - metabolism coupling ; rather the interneuron milieu is capable of generating a significant stimulus even if the endpoint is a decrease in the frequency of action potentials [ 15 , 16 ] .
there are several molecules that investigators have focused on as possible links between neuronal activity and the regulation of cerebral blood flow .
all are known to increase with synaptic transmission , either because they are involved in the process itself ( in the case of k+ and h+ ) or because they are known metabolites ( adenosine ) .
potassium and hydrogen ions are produced by synaptic transmission , and it has been shown that elevation of these ions stimulates vasodilation [ 17 , 18 ] , thus providing a possible mechanism for neurovascular coupling .
potassium channels have been found in vascular smooth muscle and have been implicated in the effect of both k and h elevations . in addition , atp - sensitive k - channels have been found in vascular smooth muscle , suggesting a direct link between neuronal activity and cerebral blood flow .
the well - known phenomenon of co2 reactivity is mediated through the action of h on cerebral arteries , rather than co2 itself .
the transduction mechanism of h into vasodilation remains elusive , although nitric oxide ( no ) has been implicated as the vasodilatory response to hypercapnia and acidosis is attenuated by no inhibitors [ 21 , 22 ] .
metabolites are an attractive option as messengers for flow - metabolism coupling , for obvious reasons .
because hypoxia and hypoglycemia do not affect activity - induced vasodilatations , attention has focused on other by - products of neuronal activity , most notably adenosine .
extracellular levels of adenosine rise sharply with neuronal activity , and topical application of adenosine to cerebral microcirculation causes vasodilation .
furthermore , adenosine has been shown to be released in response to administration of glutamate , a major neurotransmitter in cerebral cortex .
iliff et al . showed that selective blockade of adenosine receptor ( specifically receptor 2a ) attenuated the vasodilatory response to glutamate administration .
furthermore , this blockade had no effect on either resting vessel diameter or on co2 reactivity .
thus adenosine appears to play a significant role in glutamate - induced dilation of pial arterioles .
a significant body of evidence supports the function of no in flow - metabolism coupling [ 24 , 27 , 28 ] , although the exact pathway is not completely clear . a recent study by lindauer et al
. demonstrated that the vasodilatory response to whisker deflection was blocked by administration of no inhibitors .
however , the effect was restored by administration of cyclic guanosine monophosphate ( cgmp ) or no donors , suggesting that no itself is not the relevant mediator .
it has been theorized that no acts as a permissive agent , supplying a basal amount of cgmp for other mediators ( like adenosine ) to utilize as second messengers .
a wealth of functional and histological evidence supports the existence of an extensive arborization of perivascular nerves that play a role in regulation of cerebral blood flow .
the functional unit of endothelial cells , perivascular nerves , and astrocytes has been increasingly recognized as a complex network that can be considered as a single entity rather than separate subunits . referred to as the neurovascular unit , this characterization has led to investigation into supporting the unit as a whole , rather than simply focusing on one aspect .
these nerves have diverse origins and neurotransmitters and can be broadly categorized into two categories : extrinsic and intrinsic .
three main sources of extrinsic perivascular innervation have been identified : the trigeminal ganglion , the superior cervical ganglion , and the sphenopalatine ganglion .
it has been hypothesized that the main role of the sympathetic nervous system is to offer an increased tone to maintain blood pressure below the upper limit of the autoregulatory mechanism [ 31 , 32 ] .
the parasympathetic system is felt to play a role primarily in pathological states . because of the central role the trigeminovascular system plays in pain sensation
it was later discovered that calcitonin gene - related peptide ( cgrp ) , a potent vasodilator , is released from trigeminal nerves .
it was thus theorized that the trigeminovascular system plays a role in counteracting vasoconstrictive influences .
in addition , the triptan class of migraine abortive agents act presynaptically to prevent cgrp release , thereby preventing vascular engorgement and associated headache .
as shown in figure 2 , once the blood vessel dives deep into the parenchyma and leaves the virchow - robin space , they lose their extrinsic innervation and the intrinsic innervation begins .
a new set of nerves takes over , arising both from distant pathways [ 3436 ] and local interneurons .
the majority of these nerves do not abut directly on blood microvessels themselves ; rather they connect to astrocyte foot processes .
depending on the area stimulated , an increase or decrease in cbf can be elicited .
the nucleus basalis , locus coeruleus , and raphe nucleus have all been implicated as a source for innervation of cerebral microvasculature . at present , it is not entirely clear if these neurons directly contact the microvessels or if the signal is transduced through astrocytic foot - processes .
in addition to distant subcortical pathways , local interneurons also play a role in the regulation of microvascular tone .
it has recently been shown that stimulation of gabanergic interneurons causes vasodilation of regional microvessels .
furthermore , interneurons also seem to be necessary to transduce the signal from distant subcortical regions described above .
the cerebrovascular endothelium plays a central role in the regulation of cerebral blood flow . once thought to simply be an inert antithrombotic barrier ,
the endothelium is now appreciated as a dynamic organ that acts as a physiologic bridge between the blood vessel lumen and the surrounding smooth muscle . at present , this bridge is thought of as comprising 4 main chemical systems : nitric oxide ( no ) , endothelium - derived hyperpolarization factor ( edhf ) , the eicosanoids , and the endothelins .
much of the interest in the endothelium began with the discovery of nitric oxide ( no ) as the endothelium - derived relaxing factor by furchgott and ignarro in 1988 .
no is a diffusible second messenger that activates guanylate cyclase ( gc ) , present in smooth muscle cells .
guanylate cyclast in turn synthesizes cgmp which causes smooth muscle relaxation through protein kinase g ( pkg ) activation of k channels and/or closure of voltage - gated calcium channels .
the enzyme that produces no , nitric oxide synthase ( nos ) has several isoforms .
endothelial nos ( enos ) is the isoform found in the cerebral blood vessels , specifically the endothelium [ 4345 ] .
neuronal nos ( nnos ) is the isoform found in neurons . a third isoform ,
inducible nos ( inos ) has been found in the brain under pathological situations such as hypertension or exposure to endotoxin .
immunohistochemistry has localized enos to the endothelium , yet administration of a nos inhibitor ( l - nmma ) to denuded arteries produces vasoconstriction [ 47 , 48 ] .
this provides evidence for the physiological significance of nnos , although the relative contribution of enos versus nnos to resting tone of the cerebral vasculature is not known .
despite the dominance of the no paradigm in endothelial - dependent vasodilatation , it appears that there is a second mechanism that also operates in the endothelium to cause vasodilatation .
this is based on observations that when no- and eicosanoid - based pathways are fully inhibited , further dilatory capacity remains [ 4951 ] .
similar to the story of edrf prior to the discovery of no , so - called edhf represents an as of yet unelucidated pathway that may be another diffusible molecule ( the present terminology of factor is misleading based on the present data ) .
this pathway is characterized by hyperpolarization of the vascular smooth muscle and is inhibited by k - channel blockers .
golding et al . defined edhf as a dilation pathway that requires endothelium , is distinct from eicosanoid or no pathways , dilates via hyperpolarization of vascular smooth muscle , and involves k - channel activation .
it is known that the edhf pathway begins with an increase in endothelial ca stores and ends with a decrease in smooth muscle ca stores .
how these two events are linked is a matter of continuing study . at present , there are 4 main possibilities : eicosanoids , k , gap junctions , and hydrogen peroxide .
the eicosanoids are a group of vasoactive chemical mediators that are derived from arachidonic acid .
three main enzyme systems have been identified : cyclooxygenase ( cox ) , lipoxygenase ( lox ) , and epoxygenase ( epox ) .
arachidonic acid is formed from membrane phospholipids by lipases and is used as a substrate for the aforementioned enzyme systems . it should be emphasized that these enzyme systems are not limited to the endothelium ; rather they are active in a variety of tissues , notably platelets . furthermore , some products cause vasoconstriction and others cause vasodilatation .
the differential concentrations of the enzymes and their isoforms in different locations determines the overall effect on cerebral blood flow .
although all three enzyme systems have been investigated extensively in the systemic vasculature , cox is the best understood in the cerebral vasculature at present , the role of the lox and epox systems in the cerebral vasculature is poorly understood .
three cox isoforms have been found to exist in the cerebral vasculature [ 5558 ] . of the multiple metabolites produced by cox ,
the vasodilators prostacyclin ( pgi2 ) and prostaglandin e2 ( pge2 ) are predominant in normal endothelium . while important in pathologic conditions , it appears that under normal physiology the cox system is less dominant , with the no and edhf systems described above being the predominant vasodilatation paradigms .
the vasoconstrictive cox metabolites are presently thought to be most relevant in pathological situations such as traumatic brain injury and subarachnoid hemorrhage .
the fourth major chemical system active in the endothelium is that of the endothelins .
this system is comprised of two receptors ( eta and etb ) and three ligands ( et-1 , et-2 , et-3 ) .
the effect of the ligands seems to depend on the receptor rather than the ligand .
eta receptors are found predominantly in vascular smooth muscle , are stimulated by et-1 and et-2 , and mediate vasoconstriction .
etb receptors are found predominantly within the endothelium , are stimulated by all three ligands , and mediate vasodilatation [ 61 , 62 ] .
of the three ligands , et-1 appears to play the biggest role in the regulation of cerebral blood flow .
big et-1 , or produced de novo from mrna in cerebrovascular endothelial cells . in the presence of an intact endothelium , et-1 binds to etb and causes vasodilation ; in vessels denuded of endothelium , et-1 binds to eta and causes vascoconstriction [ 65 , 66 ] .
the picture is somewhat complicated by the fact that eta receptors may be found in the endothelium and etb receptors may be found in smooth muscle [ 68 , 69 ] .
interestingly , there is evidence of a balancing paradigm between the constricting actions of et-1 and no - mediated vasodilation .
et-1 secretion has been shown to be stimulated by several molecules known to be connected with no - induced vasodilation .
however , topical administration of et receptor blockers does not result in an increase in cbf .
furthermore , the long lasting effects of the endothelins make them poorly suited for the minute to minute regulation of cbf .
it is presently thought that endothelins do not contribute in a major way to resting cbf under normal physiologic conditions , although they have been shown to play a major role in several pathological conditions , most notably cerebral ischemia and cerebral vasospasm .
astrocytes are in a unique anatomical position to effect cbf ; their processes extensively ensheath brain capillaries , thereby physically linking the cerebral microvasculature with synapses .
more recently , in vitro studies have shown astrocytes to be capable of cell - to - cell communication through gap junctions , suggesting a possible role for modulation of neuronal and vascular function
. early work on the mechanism of astrocytic involvement of cerebral blood flow regulation focused on potassium .
astrocytes were known to uptake excess extracellular potassium , and newman et al . showed that the ions were then shunted to the endfeet processes . given the location of astrocytic foot processes on the cerebral vasculature and the vasodilatory effects of k , it was theorized that this was a mechanism whereby neuronal activity was linked to cerebral vasodilation .
subsequent work by paulson showed that this theory fits the temporal relationship of k increases to vasodilation better than alternative theories involving simple potassium diffusion .
found that electrical stimulation of astrocytes resulted in an increase in intracellular ca followed by vasodilation of arterioles contacted by foot processes of that astrocyte .
this response was mimicked by administration of glutamate agonists and attenuated by administration of glutamate antagonists .
astrocytes are known to express a subtype of glutamate receptor that results in an increase in intracellular ca , thus it was inferred that astrocytic foot processes sensed the synaptic activity and then responded to that activity by inducing vasodilation of the appropriate blood vessels .
much current interest is focused on the role microvascular bed in the regulation of cerebral blood flow .
specifically , there is evidence that communication within the blood vessels themselves at the microvascular level plays a key role in the overall regulation of blood flow in the brain .
since there is a substantial increase in resistance at the arteriole level , the proximal vessels must coordinate with the microvasculature in order to ensure adequate microperfusion .
first presented in vivo evidence for the presence of coordinated vasomotor responses ( cvr ) in mouse pial arterioles , by demonstrating that the local constriction response initiated by micropipette application of uridine triphosphate could travel for 300 m or more upstream from the point of application . moreover ,
conduction could be interrupted by local vascular wall injury ( light - dye technique ) .
subsequently , dietrich et al . described conducted vasodilation in penetrating arterioles isolated from the rat brain to various vasoactive agents , including adenosine and atp .
they also suggested that endothelial cells appeared to play a key role in the conducted responses of intracerebral arterioles to potassium .
the same group also found attenuation of conducted vasodilation by oxyhemoglobin treatment , suggesting a role by vascular conduction in the ischemic conditions developed after subarachnoid hemorrhage .
pioneering studies by segal and duling [ 78 , 79 ] sparked interest in cvr as a mechanism for coordinating vasomotor responses .
a simple experimental paradigm , used by these investigators to investigate vascular conduction in peripheral vessels , is to follow the longitudinal spread of vasomotor responses in arterioles to discrete stimulation of arterioles .
thus , restricted application of vasoactive substances to arteriolar segments induces both direct effects and secondary conducted vasomotor responses [ 78 , 79 ] .
the conducted dilation response could not be explained by simple diffusion or by neural innervation and is independent of blood flow and pressure , because occlusion of an arteriole to create a sealed sac did not affect the propagated dilation .
thus , the pathway of signal conduction is located exclusively in vessel wall components ( endothelium and/or smooth muscle ) .
cx40 and cx43 have been identified in smooth muscle and endothelium , whereas cx45 has only been reported in smooth muscle [ 80 , 81 ] . because connexins have electrical and conductive properties that are different from one another [ 81 , 82 ] , selective upregulation and downregulation of different connexins , that is , the change in expression profile , likely shapes the change in vasomotor conductivity after an ischemic insult .
initial studies showed that the longitudinal spread of arteriolar response to focal agonist application resembled electrical decay along a cable , suggesting that electrical signals travel by passive spread of current between cells in the vessel wall .
however , in vivo observations of conducted vasodilation indicate that some vasomotor responses exhibit little decay [ 83 , 84 ] .
for example , hyperpolarization induced by acetylcholine in arteriolar networks of the hamster cheek pouch in vivo was maintained , and even grew , as it traveled .
the mechanism for generating the additional current to aid the spread of hyperpolarization may involve inward rectifier kchannels [ 85 , 86 ] and/or na / k atpase in smooth muscle cells . in cerebral penetrating arterioles , barium chloride ( bacl2 ) attenuated
both conducted dilation and constriction responses to k , suggesting that kir may be involved in cvr in the cerebrovasculature as well .
the regulation of blood flow in the brain is exceedingly complex and only beginning to be elucidated .
we have attempted to broadly outline the major categories of mechanisms discovered so far , focusing on pressure autoregulation , metabolic regulation , and neurogenic regulation .
central to all three groups is the neurovascular unit , composed of endothelial cells , neurons , and astrocytes .
the role of this substrate in a variety of pathologic states characterized in part by failure of cerebral blood flow control ( stroke , traumatic brain injury , hypertension , alzheimer 's disease ) is the target of intense investigation and underscores the need to explore the control of blood flow at level of the microcirculation as well . | the control of cerebral blood flow is complex , and only beginning to be elucidated .
studies have identified three key regulatory paradigms .
the first is cerebral pressure autoregulation , which maintains a constant flow in the face of changing cerebral perfusion pressure .
flow - metabolism coupling refers to the brains ability to vary blood flow to match metabolic activity .
an extensive arborization of perivascular nerves also serves to modulate cerebral blood flow , so - called neurogenic regulation .
central to these three paradigms are two cell types : endothelium and astrocytes .
the endothelium produces several vasoactive factors that are germane to the regulation of cerebral blood flow : nitric oxide , endothelium - dependent hyperpolarization factor , the eicosanoids , and the endothelins .
astrocytic foot processes directly abut the blood vessels , and play a key role in regulation of cerebral blood flow .
lastly , new research has been investigating cell - cell communication at the microvascular level .
several lines of evidence point to the ability of the larger proximal vessels to coordinate vasomotor responses downstream . |
during the years 20052007 , there was a major outbreak at uppsala university hospital caused by a multi - resistant klebsiella pneumoniae strain producing ctx - m-15 ( 10 ) .
while searching for possible sources , it was noticed that the outbreak strain , like many other extended - spectrum -lactamase ( esbl)-producers ( 11 ) had a high ability to survive on different types of fabrics .
there was concern that the current laundering process at the hospital and in homes for senior citizens could be insufficient .
, all washing and drying processes were carried out at two professional laundries with years of experience of hospital laundering .
the laundering was initially planned to take place in a single professional laundry , but due to technical problems related to the lowered washing temperature ( from 70c to 60c ) , another laundry was engaged to complete the study .
the preparation of the contaminated cloth samples and the control of the level of decontamination after different procedures were performed at the department of clinical microbiology , uppsala university hospital , uppsala , sweden .
two bacterial strains were included in the study : the outbreak strain k. pneumoniae ccug54718 and enterococcus faecium nctc7171/ccug33573 .
both species are common causes of healthcare - associated infections . the relatively thermo tolerant e. faecium was also included since it is frequently used as a bioindicator in laundry studies and in european tests for determining basic bactericidal disinfectant efficiency ( 1216 ) .
prior to the experiments , a single colony from an overnight culture of each strain was inoculated in brain heart infusion broth ( bbl , becton , dickinson and co. , sparks , usa ) and incubated at 35c for 5 h to obtain a bacterial concentration of 1010 cfu / ml .
the bacterial suspension was diluted serial 1/100 with phosphate - buffered saline ( pbs ) , and from each dilution 10 l and 100 l were spread in duplicates on blood agar ( neogen , lansing , mi , usa ) .
after incubating for 20 h at 35c , viable counts were performed , using three different plates containing 30300 cfu .
tubes containing 1 ml of 5-h broth cultures of e. faecium or k. pneumoniae were exposed in duplicates to two different temperatures ( 65c and 85c ) in a thermo - block for up to 30 min .
viable counts were performed after 0 , 10 , 20 , and 30 min . as control ,
the experiment was repeated for the e. faecium strain with a broader temperature range ( 60c90c ) . in each experiment , 14 pieces of sterilized cloth ( cotton / polyester 50/50 , originating from hospital staff clothes and measuring 1010 cm ) were used .
each test sample was allowed to dry at 35c in sterile plastic petri dishes ( diameter 14 cm ) .
the remaining 12 test samples were left in the petri dishes , which were sealed with tape and placed in plastic bags for transport to the laundry .
flow chart showing how the test samples were processed at professional laundry i ( 70c ) and professional laundry ii ( 60c ) .
at arrival ,
test samples 34 stayed packaged ; they were controls and did not pass through the washing process , consisting of washing , rinsing and spinning phases in a continuous batch ( tunnel ) washer . in experiments 110 out of 13 ,
test samples 514 were put into separate laundry bags and washed the same day at 70c .
after the washing and drying by tumbling , the test samples were placed in separate clean plastic bags . in experiments 1113 ,
the test pieces were handled in the same way with one important exception : test samples 59 were excluded from the drying process .
the temperature inside the refrigerated transport box was measured when it arrived to the laboratory . within 20 h after completion of the laundering ,
the stomacher plastic bags were filled with 50 ml peptone water ( neogen , lansing , michigan , usa ) and processed in the stomacher 400 ( circulator , seward medical , uk ) for 5 min .
the bottles were shaken at 240 rpm for 10 min on a 4010 multi - tube vortexer , corning . from each suspension , 10 l , 100 l , and 2200 l were cultured on blood agar plates for the quantification of growing bacteria and e. faecium in particular .
the remaining suspension was filtered through a 0.22-m cellulose filter ( millipore corporation , bedford , usa ) , which was placed on a blood agar plate .
the plate was incubated at 37c for 48 h , and the total numbers of bacteria and of e. faecium were counted .
the cellulose filter was thereafter transferred to a plate selective for enterococci ( enterococcosel agar , becton , dickinson and company , sparks , usa ) and incubated overnight at 37c for identification of the inoculums strain .
after the first 13 experiments , 10 more followed . in these latter 10 experiments , three changes were made : the laundering process took place at another professional laundry , the temperature was lowered to 60c , and the tumble drying process was excluded for test samples 59 in all experiments .
during the years 20052007 , there was a major outbreak at uppsala university hospital caused by a multi - resistant klebsiella pneumoniae strain producing ctx - m-15 ( 10 ) .
while searching for possible sources , it was noticed that the outbreak strain , like many other extended - spectrum -lactamase ( esbl)-producers ( 11 ) had a high ability to survive on different types of fabrics .
there was concern that the current laundering process at the hospital and in homes for senior citizens could be insufficient .
, all washing and drying processes were carried out at two professional laundries with years of experience of hospital laundering .
the laundering was initially planned to take place in a single professional laundry , but due to technical problems related to the lowered washing temperature ( from 70c to 60c ) , another laundry was engaged to complete the study .
the preparation of the contaminated cloth samples and the control of the level of decontamination after different procedures were performed at the department of clinical microbiology , uppsala university hospital , uppsala , sweden .
two bacterial strains were included in the study : the outbreak strain k. pneumoniae ccug54718 and enterococcus faecium nctc7171/ccug33573 .
the relatively thermo tolerant e. faecium was also included since it is frequently used as a bioindicator in laundry studies and in european tests for determining basic bactericidal disinfectant efficiency ( 1216 ) . prior to the experiments , a single colony from an overnight culture of each strain was inoculated in brain heart infusion broth ( bbl , becton , dickinson and co. , sparks , usa ) and incubated at 35c for 5 h to obtain a bacterial concentration of 1010 cfu / ml .
the bacterial suspension was diluted serial 1/100 with phosphate - buffered saline ( pbs ) , and from each dilution 10 l and 100 l were spread in duplicates on blood agar ( neogen , lansing , mi , usa ) . after incubating for 20 h at 35c ,
tubes containing 1 ml of 5-h broth cultures of e. faecium or k. pneumoniae were exposed in duplicates to two different temperatures ( 65c and 85c ) in a thermo - block for up to 30 min .
viable counts were performed after 0 , 10 , 20 , and 30 min . as control ,
the experiment was repeated for the e. faecium strain with a broader temperature range ( 60c90c ) .
in each experiment , 14 pieces of sterilized cloth ( cotton / polyester 50/50 , originating from hospital staff clothes and measuring 1010 cm ) were used .
each test sample was allowed to dry at 35c in sterile plastic petri dishes ( diameter 14 cm ) .
the remaining 12 test samples were left in the petri dishes , which were sealed with tape and placed in plastic bags for transport to the laundry .
flow chart showing how the test samples were processed at professional laundry i ( 70c ) and professional laundry ii ( 60c ) .
at arrival ,
test samples 34 stayed packaged ; they were controls and did not pass through the washing process , consisting of washing , rinsing and spinning phases in a continuous batch ( tunnel ) washer . in experiments 110 out of 13 ,
test samples 514 were put into separate laundry bags and washed the same day at 70c .
after the washing and drying by tumbling , the test samples were placed in separate clean plastic bags . in experiments 1113 ,
the test pieces were handled in the same way with one important exception : test samples 59 were excluded from the drying process .
the temperature inside the refrigerated transport box was measured when it arrived to the laboratory . within 20 h after completion of the laundering ,
the stomacher plastic bags were filled with 50 ml peptone water ( neogen , lansing , michigan , usa ) and processed in the stomacher 400 ( circulator , seward medical , uk ) for 5 min .
the bottles were shaken at 240 rpm for 10 min on a 4010 multi - tube vortexer , corning . from each suspension , 10 l , 100 l , and 2200 l were cultured on blood agar plates for the quantification of growing bacteria and e. faecium in particular .
the remaining suspension was filtered through a 0.22-m cellulose filter ( millipore corporation , bedford , usa ) , which was placed on a blood agar plate .
the plate was incubated at 37c for 48 h , and the total numbers of bacteria and of e. faecium were counted .
the cellulose filter was thereafter transferred to a plate selective for enterococci ( enterococcosel agar , becton , dickinson and company , sparks , usa ) and incubated overnight at 37c for identification of the inoculums strain .
after the first 13 experiments , 10 more followed . in these latter 10 experiments , three changes were made : the laundering process took place at another professional laundry , the temperature was lowered to 60c , and the tumble drying process was excluded for test samples 59 in all experiments .
results of the heat tolerance experiment showed that there was a reduction of k. pneumoniae ccug 54718 with 47 log10 cfu after 30 min exposure to temperatures between 65c and 85c .
e. faecium nctc7171 exhibited a thermotolerance that was similar to that of k. pneumoniae ccug 54718 ( see fig .
2a ) . to reduce the risks of spreading an epidemic strain , the susceptible enterocccal strain , and not the multi - resistant k. pneumoniae strain , was used in all the following laundering experiments .
( a ) tolerance of strains k. pneumoniae ccug54718 and e. faecium nctc7171/ccug33573 to the temperatures 65c and 85c .
the enterococcal strain showed that the lower the temperature , the longer time of exposure was needed for reducing the bacterial number . at temperatures
70c , the major killing took place during the first 10 min ( see fig .
all test samples were impregnated with an average of 1010 cfu e. faecium ( test samples 12 ) .
unwashed test samples ( 34 )
contained 49 log10 cfu when returned to the laboratory .
the median washing time at 70c was 15 min ( range 1020 min ) and for the full washing circle 30 min ( range 3055 min ) .
the washing process followed by tumble drying , reduced the bacterial numbers with up to 9 log10 cfu .
the observed killing effect of the tumble drying , was the background to the changes in the study protocol ; in the following experiments the washing process and the tumble drying was separated at all times ( see fig .
3 ) . the median drying temperature during tumbling was 78c ( range 63c85c ) , and the drying process lasted for a median of 22 min ( range 1730 min ) .
viable counts from test samples before and after a full laundry process , using 70c wash cycle and tumbling at a median temperature of 78c .
each point in the graph represents the median number of bacterial cells from two samples before washing and 10 samples after the tumbling .
the test samples were impregnated with the same level of bacteria as in the first 13 experiments ( average of 1010 cfu ) , but the unwashed test samples ( 34 ) contained essentially the same amount of bacteria as test samples 12 at the return to the laboratory .
the median washing time at 60c was 15 min ( range 1317 min ) and for the full washing circle 30 min ( range 3033 min ) .
the corresponding figure for the tumble drying was 34 log10 cfu , yielding a total reduction of bacteria of up to 9 log10 cfu for a full laundering process including the tumble drying ( see fig .
viable counts from test samples before the laundering , after a wash cycle at 60c , and after a full laundry process ( washing at 60c and tumble drying at a median temperature of 117.5c ) .
each point in the graph represents the median number of bacterial cells from two samples before washing and five samples after washing without tumble drying and five samples after washing with tumble drying .
the median temperature during tumble drying was 117.5c ( range 112c123c ) , whereas the total time for drying was a median of 14.8 min ( range 1217.4 min ) .
the temperature during the tumble drying was thereby higher but lasted for a shorter time period than during the first 13 experiments .
the number of e. faecium cells was not possible to count in the first two experiments due to overgrowth of non - fermenting gram - negative bacteria .
results of the heat tolerance experiment showed that there was a reduction of k. pneumoniae ccug 54718 with 47 log10 cfu after 30 min exposure to temperatures between 65c and 85c .
e. faecium nctc7171 exhibited a thermotolerance that was similar to that of k. pneumoniae ccug 54718 ( see fig .
2a ) . to reduce the risks of spreading an epidemic strain , the susceptible enterocccal strain , and not the multi - resistant k. pneumoniae strain , was used in all the following laundering experiments .
( a ) tolerance of strains k. pneumoniae ccug54718 and e. faecium nctc7171/ccug33573 to the temperatures 65c and 85c .
the enterococcal strain showed that the lower the temperature , the longer time of exposure was needed for reducing the bacterial number . at temperatures
70c , the major killing took place during the first 10 min ( see fig .
all test samples were impregnated with an average of 1010 cfu e. faecium ( test samples 12 ) .
unwashed test samples ( 34 )
contained 49 log10 cfu when returned to the laboratory .
the median washing time at 70c was 15 min ( range 1020 min ) and for the full washing circle 30 min ( range 3055 min ) .
the washing process alone reduced the number of bacteria with about 5 log10 cfu . the washing process followed by tumble drying , reduced the bacterial numbers with up to 9 log10 cfu .
the observed killing effect of the tumble drying , was the background to the changes in the study protocol ; in the following experiments the washing process and the tumble drying was separated at all times ( see fig .
the median drying temperature during tumbling was 78c ( range 63c85c ) , and the drying process lasted for a median of 22 min ( range 1730 min ) .
viable counts from test samples before and after a full laundry process , using 70c wash cycle and tumbling at a median temperature of 78c .
each point in the graph represents the median number of bacterial cells from two samples before washing and 10 samples after the tumbling .
the test samples were impregnated with the same level of bacteria as in the first 13 experiments ( average of 1010 cfu ) , but the unwashed test samples ( 34 ) contained essentially the same amount of bacteria as test samples 12 at the return to the laboratory .
the median washing time at 60c was 15 min ( range 1317 min ) and for the full washing circle 30 min ( range 3033 min ) .
the corresponding figure for the tumble drying was 34 log10 cfu , yielding a total reduction of bacteria of up to 9 log10 cfu for a full laundering process including the tumble drying ( see fig .
viable counts from test samples before the laundering , after a wash cycle at 60c , and after a full laundry process ( washing at 60c and tumble drying at a median temperature of 117.5c ) .
each point in the graph represents the median number of bacterial cells from two samples before washing and five samples after washing without tumble drying and five samples after washing with tumble drying .
the median temperature during tumble drying was 117.5c ( range 112c123c ) , whereas the total time for drying was a median of 14.8 min ( range 1217.4 min ) .
the temperature during the tumble drying was thereby higher but lasted for a shorter time period than during the first 13 experiments .
the number of e. faecium cells was not possible to count in the first two experiments due to overgrowth of non - fermenting gram - negative bacteria .
in the present study , a simple method for simulating a full laundering process in a professional laundry was developed to evaluate how different washing temperatures affected the bacterial decontamination .
the results showed that the decontamination of the test samples was comparable at 70 and 60c .
furthermore , it became evident that the following drying process made an important contribution to the reduction of the bacterial number .
in fact , without tumble drying there was an obvious risk of contaminating the test samples heavily with non - fermenting gram - negative bacteria during the washing process .
other studies have yielded similar results ( 17 , 18 ) . in recent years , the interest in laundering processes and their ability to reduce microbial contamination has mounted .
the basis for this is to a large extent the increased frequency of outbreaks caused by multi - resistant bacteria in both healthcare facilities and in the community . during outbreak investigations ,
infection control teams usually check the laundry routines , especially if an outbreak does not seem to have an obvious cause .
studies dealing with laundry have yielded a wide range of results , and they are not always easy to interpret or draw conclusions from .
this is to a large extent due to the high number of variables that can affect the outcome , that is , the type of microorganisms / washing machine / fabric , inoculum concentration , laundering conditions , added detergents and/or biocides , drying routines , etc .
some studies have focused on the washing temperature , whereas others have included the drying temperature or the energy costs ( 13 ) .
however , tumble drying the textiles has in several studies , including this , been shown to have significant impact on the decontamination ( 17 ) .
ironing also seems to be of importance for reducing the bacterial number ( 18 , 20 ) .
the role of procedures like tumble drying and ironing , which follow the washing cycle , probably increases when the laundry process can not be fully monitored .
this is usually the case when conventional washing machines are used in old people 's homes , other types of healthcare facilities and under home - like conditions .
the temperature in these instances is frequently lowered to 3040c to not damage the clothes . at these temperatures , the risk of laundry malodor and contamination is obvious ( 2123 ) . to let the hospital staff wash their hospital clothing in their private washing machines without tumble
enterococci are notoriously difficult to get rid of and are therefore often used as bioindicators ( 1315 ) .
previous studies have shown that they can survive laundering at 60c for 10 min ( 1213 ) but not 75c ( 24 ) .
their high tolerance to heat was also shown in the present study by e. faecium strain nctc7171 .
there was , however , a difference between the tolerance at temperatures below 70c and those at 70c or more . in the former case ,
this difference in temperature tolerance was not noticed when laundering at 70c and 60c , indicating that not only the washing temperature but also the centrifugation and rinsing contributes to the decontamination in a washing process .
the multi - resistant k. pneumoniae strain , which caused the major outbreak at the hospital , was surprisingly heat tolerant .
due to the risks of further dissemination , it was not used during the laundering experiments .
it can be argued that a susceptible member of the enterobacteriaceae family should have been included in the laundering experiments instead .
they seem , however , to be not as good environmental survivors as their multi - resistant variants ( 25 ) , and there was a need for rapid answers with a limited budget . a more comprehensive study , including also susceptible k. pneumoniae strains , viruses and fungi , was therefore not possible .
when starting up a new method , there are often problems . one of them was the transportation of samples to and from the professional laundry , which was not optimal during the first part of the study ( when washing at 70c ) .
furthermore , the laboratory technician who performed the initial experiments was not accustomed to dealing with this type of sample or carrying out viable counts .
a combination of these factors is probably the cause of the large variation in bacterial numbers during the laundering at 70c .
a method for simulating a full laundering process in a professional laundry was developed in this study .
when applied , the method showed that it is possible to maintain sufficient textile hygiene where bacteria are concerned when washing at 60c without adding biocides .
however , for the best result the washing cycle must be followed by tumble drying .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | backgroundseveral major outbreaks in healthcare facilities have occurred with the emergence of multi - resistant bacteria .
a possible route for dissemination is the hospital textiles and inadequate laundering of them .
the aim of this study was to develop an easy - to - use method for simulating the laundering process of hospital textiles , and thereafter apply the method when evaluating the decontaminating efficacy of two different washing temperatures.methodsthe laundering process , including tumble drying , took place at two professional laundries .
enterococcus faecium was used as bioindicator.resultsthe results showed that a lowering of the washing temperature from 70c to 60c did not affect the decontamination efficacy ; the washing cycle alone reduced the number of bacteria with 35 log10 cfu , whereas the following tumble drying reduced the bacterial numbers with another 34 log10 cfu , yielding the same final result independent of washing temperature . without tumble
drying , there was an obvious risk of adding non - fermenting gram - negative bacteria to the fabric .
these bacteria originated from the washing cycle.conclusiona simple method to simulate hospital laundering was developed . to save energy , it is possible to use a washing temperature of 60c , but the washing cycle should be followed by tumble drying , and the whole laundering process needs to be monitored to maintain sufficient textile hygiene . |
dner kebab is one of the most popular and consumed ethnic ready - to - eat ( rte ) dishes in europe .
people are used to eating this takeaway food as a meal substitute or as a snack in several different occasions depending also on the local customs and traditions . according to a survey conducted for unaitalia ,
the kebab is the favorite dish for almost 30% of young people under 34 years old ( colussi , 2014 ) .
the rapid spread of this dish is mainly due to its widespread distribution and selling network , consisting of shops , kiosks and stands open several hours a day to sell without competition from other kinds of restaurant ( paolini , 2005 ) .
this has certainly had an impact on sales with nearly 1.3 million portions of dner kebab , as an example , being sold by static and mobile vendors every day in the united kingdom ( british kebab awards , 2013 ) .
moreover , the statistics highlight that in italy since 2001 there has been a continuous rising in the percentage of people consuming at least one meal outside homes ( cersosimo , 2011 ) .
there is not a single definition of dner kebab , there has been an evolution of the recipe as well as of the terminology in every geographical area . with regard to the etymology , it can be stated that the word kebab means roasted meat ( from the arabian kabab ) and that is usually preceded by an adjective which allows identifying the dish or the method of cooking meat ( in this case dner refers to the vertical rotating skewer ) . using a generic description
, we can say that dner kebab is a traditional turkish dish ( the inventor seems to be a turk emigrated to germany ) consisting of meat cooked on a vertical rotating spit and portioned at the time of consumption or ordering .
the meat of the original recipe can be lamb , mutton , beef , goat or chicken .
the method of preparation consists of stacking seasoned or marinated slices of lean meat on a vertical spit , giving a cylindrical shape to the finished product . nowadays
the meat used for the recipes comes from big companies that export frozen skewers made mostly of chicken , turkey and beef . at the shops , the meat cooking process by radiant infrared heat emanated from hot plates or gas - powered burners ( reaching a temperature between 200 and 300c )
starts when the shop opens and goes on until the meat is cut for consumption . at this point
thin slices of cooked meat are cut and put in a pocket of pita bread or in a piadina .
this dish is often accompanied by a choice of sauces ( mayonnaise , ketchup , tzatziki , harissa ) and vegetables ( heinz and hautzinger , 2007 ) .
nevertheless , in most of the shops an industrial product often made with poor quality meat is served ( castellani , 2007 ) .
the studies relating to the nutritional aspects of this preparation are very few , and most of them are limited to the analysis of only two ingredients ( bread and meat ) or only meat ( marletta et al . , 2010 ; vazgecer et al . ,
commissioned by the british government , its scope was to verify the correspondence of the information contained in the label of the kebab and its real content ( animal species identification ) and to analyze the nutritional composition of the kebab , mainly bread and meat , creating a comparison with the nutritional guidelines and indications ( lacors , 2009 ) .
the purpose of the present study was to evaluate the variability of the recipe and the nutritional quality ( through a detailed analysis of the nutrients ) of an ethnic dish distributed in two towns of veneto region and prepared according to a commercial formula by mixing meat of dner , bread , vegetables and sauces .
it must be said that the choice of the two towns is not completely random .
verona is the fifth italian town for inflow of foreign tourists ( 1,800,000 foreign guests in 2012 ; osservatorio nazionale del turismo , 2013 ) and it also has a large population of university students .
the same considerations can not be transferable to the town of vicenza that substantially relies only on local consumers .
moreover , for each nutrient fact the contribution of a full portion of dner kebab was calculated and then compared to the suggested daily reference intakes for the italian population . given that in the literature information on proximate composition and nutritional value of kebab intended as gastronomic preparation is incomplete , in this preliminary study it was therefore decided to give priority toward a most detailed analysis , investigating almost all the macro - nutrients in addition to measure the energy value , limiting the sampling rate to a number of well - defined retailers .
the determination of the number and location of the retailers to be sampled was done in collaboration with the veterinary services of the local health authority responsible for those areas .
the sampling activity , conducted between september and october 2012 , involved a total of 20 public retailers , 7 of which were in the town of verona , 11 in the town of vicenza and 2 in the town of bassano del grappa ( vi ) .
for each point of sell , one sampling time has been performed , reaching a total of 10 samples of sandwiches and 10 samples of piadinas .
each dner kebab was sampled keeping the bread ( pita or flat bread ) and the meat separated from the vegetables ( tomato , green salad , onion , pepper , cabbage , squash ) and from the sauces .
all these main ingredients were collected according to the usual proportions used in each shop but kept in three separate containers ( gas impermeable ) with the aim to prevent loss of ingredients or macronutrients between the different matrixes ( in particular to avoid migration of humidity especially from vegetables and/or sauce to bread ) . at the same time a copy of the label of the skewer was taken , so that the origin , composition and animal species of the meat could be known . after the individual weighing of each of the four ingredients and calculation of the respective percentage , a portion of each one after grinding ( 4000 rpm min for 5 sec , grindomix gm200 ; retsch , dusseldorf , germany ) was used to obtain a representative mixed sample that was homogenized again ( 4000 rpm min for 5 sec ) . the homogenate was then freeze - dried and subsequently homogenized ( 4000 rpm min for 5 sec ) to further improve the precision of the analytical data .
the residual quotes of each ingredient were used to measure collagen ( in the case of meat ) and to measure the ph value ( for all others meat included , in order to verify if their values were in line with the usual ones or were modified by technological processes ) .
after homogenization in distilled water ( 1:10 w / v , at 13,000 rpm min for 30 sec with a disperser ultraturrax t25 basic ; ika werke , staufen , germany ) , ph was measured using a portamess ph - meter ( knick 910 ; knick , berlin , germany ) equipped with inlab 427 electrode ( mettler toledo , urdof , switzerland ) .
the measurement of moisture was carried out gravimetrically by drying approximately 10 g of sample ( two decimal figures ) in a convection oven at 1032c until constant weight ( aoac 983.18 ; aoac , 1990 ) . to calculate protein , 0.5 g of sample ( four decimal figures )
were submitted to the kjeldahl procedure , total nitrogen was converted into crude protein using the 6.25 factor ( aoac 928.08 ; aoac , 1990 ) .
ashes were measured by gravimetric method after combustion of 3 g of sample ( four decimal figures ) in a muffle furnace at 5502c until obtaining a white residue ( aoac 923.03 ; aoac , 1990 ) .
the percentage of fat was determined gravimetrically ( folch et al . , 1957 ) on 5 g of sample ( two decimal figures ) .
the salt content was determined on 2.5 g of sample ( four decimal figures ) using the volhard method ( aoac , 1990 ) .
gross energy was measured by combustion of 1 g of sample ( four decimal figures ) in an excess of oxygen in a bomb calorimeter under standardized conditions ( fao , 2011 ) .
total dietary fibre was determined submitting 1 g of sample ( four decimal figures ) to enzymatic - gravimetric method ( aoac 985.29 ; aoac , 1990 ) . to measure carbohydrate ( starch ) , 0.5
g of sample ( four decimal figures ) were analysed by enzymatic method ( aoac 979.10 ; aoac , 1990 ) .
the method consisted of an initial step of acid hydrolysis ( conducted with hcl 6 m on 1 g weighed with four decimal figures warmed at 1002c for 18 h ) , followed by hydroxyproline oxidation to pyrrole by means of chloramine t ( sigma - aldrich , milan , italy ) and the registration of its light absorption at 558 nm after complexation with the ehrlich reagent ( edwards and obrien , 1980 ) . to convert the absorbance values in concentrations ,
a calibration curve was fitted using diluted concentrations of a pure standard of hydroxyproline ( sigma - aldrich ) .
to calculate the percentage of collagen , the concentration of hydroxyproline was multiplied by 8 . for fatty acid ( fa )
analysis , the anhydrous fat ( folch et al . , 1957 ) was first esterified in an acid medium then analyzed by gas chromatography .
briefly , 40 mg of fat weighed in a test tube with screw cap with teflon sealing were derivatized using 1 ml of methanolic hcl 3 n ( supelco italy , milan , italy ) at 902c for 1 h mixing the solution every 10 min .
after cooling to room temperature , 1 ml of distilled water was added and mixed , which was followed by the addition of 2 ml of n - hexane ( sigma - aldrich ) .
the mixture was centrifuged at 400xg for 10 min ( eppendorf centrifuge 5804 ; eppendorf , hamburg , germany ) and the upper phase , containing the fatty acids methyl esters ( fame ) , was transferred into a clean vial .
fatty acids methyl esters were analyzed using a gas chromatograph ( shimadzu gc-17a ; shimadzu co. , kyoto , japan ) equipped with a split - splitless injector ( set at 280c ) and a flame ionization detector set at 250c .
one l of fame mix was injected in split mode ( ratio 1:40 ) with hydrogen at 40 cm sec as carrier gas and an omegawax 250 capillary column ( 30 m , 0.25 mm , 0.25 m ; supelco inc . , bellefonte , pa , usa ) used to separate the components according to initial temperature of 40c for 5 min , first ramp at 10c min up to 120c ( holding time 0.5 min ) and second ramp at 4c min up to 240c ( holding time 5 min ) . the response factor for each peak
was determined from an equal weight commercial standard mix of fatty acids methyl esters ( sigma - aldrich ) .
the original solution was diluted to give a final concentration of 1.0 mg / ml total fame .
fatty acids methyl esters were identified by matching their retention time with that of an external standard mixture ( sigma - aldrich ) and expressed as a percentage of total fatty acids and in concentration of mg/100 g of dner kebab using the following equation ( greenfield and southgate , 2007 ) :
fatty acid ( mg/100g)=fame% x fat% x 0.945 x 10
mean and standard deviation of the data are reported in tables 1 - 3 . a one - way anova test was used to determine significant differences within two fixed factors : town of sampling ( to verify if a standardization of the recipe exists ) and median value of the meat percentage (
since the cooked meat is the more abundant component of the dish as well as with greater nutrient density it was decided to check how much the percentage of added meat can modify the quantity and quality of macronutrients .
the percentage of meat was used as fixed factor since in all the sampled points of sell the dner had the same composition in terms of animal species ) .
pearson correlation coefficients were used to explore possible relationships between the percentage of the ingredients and chemical indexes .
statistical analyses of the data were performed by spss version 15.0 ( spss inc . , chicago , il , usa ) .
the determination of the number and location of the retailers to be sampled was done in collaboration with the veterinary services of the local health authority responsible for those areas .
the sampling activity , conducted between september and october 2012 , involved a total of 20 public retailers , 7 of which were in the town of verona , 11 in the town of vicenza and 2 in the town of bassano del grappa ( vi ) .
for each point of sell , one sampling time has been performed , reaching a total of 10 samples of sandwiches and 10 samples of piadinas .
each dner kebab was sampled keeping the bread ( pita or flat bread ) and the meat separated from the vegetables ( tomato , green salad , onion , pepper , cabbage , squash ) and from the sauces .
all these main ingredients were collected according to the usual proportions used in each shop but kept in three separate containers ( gas impermeable ) with the aim to prevent loss of ingredients or macronutrients between the different matrixes ( in particular to avoid migration of humidity especially from vegetables and/or sauce to bread ) . at the same time a copy of the label of the skewer was taken , so that the origin , composition and animal species of the meat could be known . after the individual weighing of each of the four ingredients and calculation of the respective percentage , a portion of each one after grinding ( 4000 rpm min for 5 sec , grindomix gm200 ; retsch , dusseldorf , germany ) was used to obtain a representative mixed sample that was homogenized again ( 4000 rpm min for 5 sec ) . the homogenate was then freeze - dried and subsequently homogenized ( 4000 rpm min for 5 sec ) to further improve the precision of the analytical data .
the residual quotes of each ingredient were used to measure collagen ( in the case of meat ) and to measure the ph value ( for all others meat included , in order to verify if their values were in line with the usual ones or were modified by technological processes ) .
after homogenization in distilled water ( 1:10 w / v , at 13,000 rpm min for 30 sec with a disperser ultraturrax t25 basic ; ika werke , staufen , germany ) , ph was measured using a portamess ph - meter ( knick 910 ; knick , berlin , germany ) equipped with inlab 427 electrode ( mettler toledo , urdof , switzerland ) .
the measurement of moisture was carried out gravimetrically by drying approximately 10 g of sample ( two decimal figures ) in a convection oven at 1032c until constant weight ( aoac 983.18 ; aoac , 1990 ) . to calculate protein , 0.5 g of sample ( four decimal figures )
were submitted to the kjeldahl procedure , total nitrogen was converted into crude protein using the 6.25 factor ( aoac 928.08 ; aoac , 1990 ) .
ashes were measured by gravimetric method after combustion of 3 g of sample ( four decimal figures ) in a muffle furnace at 5502c until obtaining a white residue ( aoac 923.03 ; aoac , 1990 ) .
the percentage of fat was determined gravimetrically ( folch et al . , 1957 ) on 5 g of sample ( two decimal figures ) .
the salt content was determined on 2.5 g of sample ( four decimal figures ) using the volhard method ( aoac , 1990 ) .
gross energy was measured by combustion of 1 g of sample ( four decimal figures ) in an excess of oxygen in a bomb calorimeter under standardized conditions ( fao , 2011 ) .
total dietary fibre was determined submitting 1 g of sample ( four decimal figures ) to enzymatic - gravimetric method ( aoac 985.29 ; aoac , 1990 ) . to measure carbohydrate ( starch ) , 0.5
g of sample ( four decimal figures ) were analysed by enzymatic method ( aoac 979.10 ; aoac , 1990 ) .
the method consisted of an initial step of acid hydrolysis ( conducted with hcl 6 m on 1 g weighed with four decimal figures warmed at 1002c for 18 h ) , followed by hydroxyproline oxidation to pyrrole by means of chloramine t ( sigma - aldrich , milan , italy ) and the registration of its light absorption at 558 nm after complexation with the ehrlich reagent ( edwards and obrien , 1980 ) . to convert the absorbance values in concentrations ,
a calibration curve was fitted using diluted concentrations of a pure standard of hydroxyproline ( sigma - aldrich ) . to calculate the percentage of collagen , the concentration of hydroxyproline was multiplied by 8 . for fatty acid ( fa )
briefly , 40 mg of fat weighed in a test tube with screw cap with teflon sealing were derivatized using 1 ml of methanolic hcl 3 n ( supelco italy , milan , italy ) at 902c for 1 h mixing the solution every 10 min .
after cooling to room temperature , 1 ml of distilled water was added and mixed , which was followed by the addition of 2 ml of n - hexane ( sigma - aldrich ) .
the mixture was centrifuged at 400xg for 10 min ( eppendorf centrifuge 5804 ; eppendorf , hamburg , germany ) and the upper phase , containing the fatty acids methyl esters ( fame ) , was transferred into a clean vial .
fatty acids methyl esters were analyzed using a gas chromatograph ( shimadzu gc-17a ; shimadzu co. , kyoto , japan ) equipped with a split - splitless injector ( set at 280c ) and a flame ionization detector set at 250c .
one l of fame mix was injected in split mode ( ratio 1:40 ) with hydrogen at 40 cm sec as carrier gas and an omegawax 250 capillary column ( 30 m , 0.25 mm , 0.25 m ; supelco inc . , bellefonte , pa , usa ) used to separate the components according to initial temperature of 40c for 5 min , first ramp at 10c min up to 120c ( holding time 0.5 min ) and second ramp at 4c min up to 240c ( holding time 5 min ) . the response factor for each peak
was determined from an equal weight commercial standard mix of fatty acids methyl esters ( sigma - aldrich ) .
the original solution was diluted to give a final concentration of 1.0 mg / ml total fame .
fatty acids methyl esters were identified by matching their retention time with that of an external standard mixture ( sigma - aldrich ) and expressed as a percentage of total fatty acids and in concentration of mg/100 g of dner kebab using the following equation ( greenfield and southgate , 2007 ) :
fatty acid ( mg/100g)=fame% x fat% x 0.945 x 10
a one - way anova test was used to determine significant differences within two fixed factors : town of sampling ( to verify if a standardization of the recipe exists ) and median value of the meat percentage ( since the cooked meat is the more abundant component of the dish as well as with greater nutrient density it was decided to check how much the percentage of added meat can modify the quantity and quality of macronutrients .
the percentage of meat was used as fixed factor since in all the sampled points of sell the dner had the same composition in terms of animal species ) .
pearson correlation coefficients were used to explore possible relationships between the percentage of the ingredients and chemical indexes .
statistical analyses of the data were performed by spss version 15.0 ( spss inc . , chicago , il , usa ) .
from the measures obtained some considerations about the composition of a medium portion of dner kebab can be drawn .
looking at the origin of the ingredients , it is evident that the noticeable differences among different preparations are mainly consequence of the quantity of ingredients used .
in fact , from the information collected at the point of sell it was evident that bread and sauces are usually bought at supermarkets , and so they are made with the same ingredients ; vegetables are bought from the same supplier ; meat comes from the same producers , meaning that animal species , additives and preparation are the same for each skewer . in all the sampling point the meat was chicken or a chicken / turkey mixture .
the predominant ingredient was certainly meat ( percentage ranged between 34.7 and 39.5% ) in both towns .
with regard to bread , vegetables and sauces the amount present did not vary significantly between the recipes of the two towns .
detailed percentages of the ingredients and the significance of the data obtained are reported in table 1 .
the ph value of the individual ingredients ranged between a minimum of 6.57 and a maximum 7.05 , 4.88 and 6.44 , 4.39 and 5.48 , 3.81 and 4.49 respectively in meat , bread , vegetables and sauce ( table 1 ) .
considering now the variables of narrower nutritional interest , we must keep in mind that the values refer to the sample assembled , after a weighted mixing of the four components ( meat , bread , vegetables and sauce ) .
the data collected were analyzed in order to focus attention on the differences between the towns of sampling and the influence of the meat percentage on the chemical composition .
concerning the second factor , the samples were grouped according to the value of the median ( 36.7% ) of meat percentage .
comparing the two towns , the data showed a significant difference in terms of carbohydrate content , ranging from 10.1 g/100 g measured in the samples from vicenza to 13.8 g/100 g in the samples collected in verona ( table 2 ) .
this difference is probably due to the different percentage of bread used in the preparation of the dish ( r=0.747 , p<0.001 between bread percentage and carbohydrate concentration ) .
the statistically most interesting observations can be made observing the various nutritional intakes of the kebab considering a meat content above or below the median value . as can be seen from table 2 ,
samples with a higher content of meat were also those with a higher energy intake ( 237.4 kcal/100 g compared to 215.8 kcal ; r=0.595 , p<0.01 between meat percentage and energy ) , higher protein ( 14 g/100 g vs 12 g/100 g ) and fat ( 9.6 g/100 g compared to 7.8 g/100 g ) content .
another consequence of high quantity of meat is the lower content of total dietary fibre which is equal to 1 g/100 g against 1.3 g/100 g of the samples with an average content of meat lower than 36.7% ( r=0.642 , p<0.01 ; r=0.621 , p<0.01 and r=-0.55 , p<0.05 between meat percentage and protein , fat and dietary fibre content respectively ) .
salt concentration ranged between 1 and 1.1% for samples respectively below and above the value of the median of the meat percentage .
it is interesting to note that salt percentage was relatively constant and independent from the other chemical variables and ingredients .
the data of collagen percentage has been expressed on the meat , with the aim to highlight the quality of this component .
it is useful to point out that all the dner kebab sampled , according to the list of ingredients on the label of the meat , were almost all consisting of chicken or mixed chicken / turkey and came from the same producers . from the analysis of fatty acids
, it can be seen that the most represented category was that of monounsaturated fatty acids ( 2445.9 and 2678.4 mg/100 g of sample in verona and vicenza respectively ) , compared to saturated and polyunsaturated , as it can be seen in table 3 .
as expected , the concentration of fatty acids was affected by the percentage of meat in the dner kebab . in particular , significant differences were observed for saturated fatty acids ( 2638.3 and 1959.9 mg/100 g in the samples with a content of meat respectively above and below 36.7% ) and monounsaturated ( 3006.1 mg/100 g above the median value and 2188 mg/100 g below ) .
instead , the amount of polyunsaturated was not clearly influenced neither by the percentage of meat nor from that of the other ingredients used for the preparation of the dish .
the most represented fatty acid was oleic acid ( 18:1n9 ) , with values of 2245.6 and 1947.9 mg/100 g in vicenza and verona respectively , followed by linoleic acid ( 18:2n6 ) , palmitic acid ( 16:0 ) , stearic acid ( 18:0 ) , palmitoleic acid ( 16:1n7 ) , -linolenic acid ( 18:3n3 ) , vaccenic acid ( 18:1n7 ) , arachidonic acid ( 20:4n6 ) and
it is clear in table 3 that the amount of certain fatty acids was significantly affected by the amount of meat used to prepare the dner kebab .
the main quantitative differences that have been noted concerning single compounds are relative to palmitic , stearic , palmitoleic , oleic , vaccenic and -linolenic acid . finally , the ratio omega-6/omega-3 , whose values ranged from 4.1 to 17 with an average value of 11.5 , was not affected neither by the place of sampling nor by the percentage of meat .
it is noteworthy that the preparation based on dner kebab appeared extremely standardized among different commercial retailers and also between different towns , and this fact is mostly due to the origin of the ingredients .
the majority of the skewers were imported from the same german company and in most cases the sauces used , like mayonnaise and ketchup , were bought at the supermarket .
the assumption shown in the introduction for the existence of any differences in the formulation of the dish between the two towns to satisfy a different type of consumer ( occasional vs habitual consumer ) can be refuted , even if these are preliminary data .
the ph of the meat was high if compared with what may be the common values for fresh breast poultry meat ( yl - ajos et al . , 2007 ) .
the use of polyphosphates and the industrial storage in regime of freezing of the skewer before commercialization to retailers , in addition to cooking at the point of sell , may cause a significant rise in the ph value ( ergnl and kundakci , 2007 ; demirok et al . , 2011 ) .
in contrast , the low ph values of the sauces could be explained by the frequent use of yoghurt for their preparation . for a more complete discussion about the nutritional intake arising from the dner kebab ,
the data obtained from the present study were compared with the rdas ( set of reference values for the diet in the population and for healthy people ) published by the italian society of human nutrition in 2012 .
the reference values used were the recommended intake for the population or nutritional objective for the prevention or reference interval for the intake of macronutrients or level of adequate intake with regard to protein , saturated fat , carbohydrates , fibre and lipids or tolerable levels of intake for the italian population in the case of salt ( sinu , 2012 ) .
considering an individual with a low to medium level of physical activity , as it may be that of an office worker , the energy needs for a male are of 2500 kcal / day , while for a female is 2000 kcal / day . comparing these needs with the mean value of 226.125.7 kcal/100 g of a mean serving portion of dner kebab ( 402 g for a total of 900.9 kcal ; table 4 )
, it can be noted that the contribution of this rte is equal to 36.0 and 45.0% of the daily energy requirements for males and females respectively ( table 4 ) .
the energy was correlated with the percentage of meat , as previously said , whereas the contribution of vegetables and sauces used in the preparation was quite marginal ( r=-0.57 , p<0.01 ; r=-0.44 p<0.05 ) .
the protein content of a portion of dner kebab ( 12.952.12 g/100 g equal to 51.7 g for a mean serving size ) can be considered equal to 82.1% of the reference interval ( ri ) for male and 95.7% for female . for total carbohydrates
( mainly represented by starch ) , it can be seen that the values obtained ( 11.33.7 g/100 g equal to 43.8 g for a serving size ) fall below the ri ( 4560% of energy ) .
looking at the lipid intake , a meal based on dner kebab ( 34.7 g per serving size ) can cover , on average , 41.8 and 51.8% of the daily ri of fat for male and female respectively . in comparison with others studies ,
the mean energy content ( 226.1 kcal/100 g ) was lower than 268 kcal/100 g ( marletta et al . , 2010 ) or that of 336 kcal/100 g registered by the lacors study .
however , it must be pointed out that in both studies the values were indicative solely of bread plus meat or only meat .
the low values found in this study , where the total ingredients were analyzed after pooling , may be due to the low energy density of the vegetables but also to the use of meat with relatively low lipid content .
the lipids content , expressed per 100 g of sample , was 8.6 , 16.7 and 20.6 g respectively for the present study , ( marletta et al . , 2010 ) and lacors .
saturated fat was 2.3 , 6 and 9.8 g. such differences are certainly determined by the different sampling approach , but also by the quality and kind of meat species ( in particular with reference to saturated fat ) . in the kebabs collected for the lacors study , more than 70% of the meat was ovine and/or beef , while in the present study the meat was poultry or poultry / turkey mixture .
the protein content , that was obviously affected by the sampling approach in relation to the amount of meat , was 29.7% ( marletta et al .
, 2010 ) , 18.7% ( lacors , 2009 ) and 12.95% in the present study .
the collagen concentration ( a component of the total protein ) normally is less than 1% in chicken and turkey breast , and slightly greater than 1% in the case of leg muscles ( food standard agency , 2004 ) .
however , the value increases as a function of the amount of skin that is inserted in the dner preparation which means that the values measured in the present study may appear high but are still lower than those shown by vazgecer et al .
in other words , it means that a serving size of dner kebab in both studies covers , if not completely , most of the recommended daily quota for sodium chloride with severe concerns in the case of people that suffer for hypertensive phenomena or more in general for the prevention of cardiovascular disease ( starmans - kool et al . , 2011 ) .
with regard to carbohydrates and dietetic fibre , the contribution arising from dner kebab to these nutrients appeared to be less than 20% of the recommended values .
carbohydrates for serving was equal to 15.5% ( males ) and 19.5% ( females ) whereas fibre touches 15% ( slightly less for males ) of the suggested reference daily intake .
on the other hand , the protein content of a serving of dner kebab ( 51.7 g ) is enough to cover almost the full daily requirements for females whereas it reaches 82.1% in the case of males .
kebab with yoghurt a turkish traditional food made of mutton , veal , flat bread , whole fat yoghurt , butter , tomato paste , green pepper , red tomato and tap water - has a chemical composition of 226 kcal/100 g , moisture 60.4% , protein 9.2% , fat 15.6% , ash 1.6% , salt 0.95% , fibre 1.1% , carbohydrate 12.07% ( biringen lker et al . , 2013 ) .
therefore , it is a kebab preparation with a chemical composition very similar to that of the dner kebab sampled in the towns of verona and vicenza .
looking at the fats , from the point of view of nutritional quality , consuming a serving of dner kebab provides an intake of saturated fatty acids ( the most harmful to health ) equal to 33.4% and 42.5% of ri respectively for males and females , whereas the coverage of n3 requirements is less than 17% .
polyunsaturated fats were 29.2 and 37.2% of daily ri for males and female respectively . among n3 fats , -linolenic acid that shows positive effects on cardiovascular health reached the average concentration of 156 mg/100 g of dner kebab which is half of the minimum value necessary to define a food a source of omega-3 fat ( reg .
on the other hand , the average ratio omega-6/omega-3 found in the samples of the present study was rather high if compared to the levels normally suggested that should be at least 4:1 or minor ( simopoulos , 2008 ) .
the preparations based on dner kebab analyzed in this study have highlighted the nutritional value and energy content of this rte food . there is a high content of protein as well as a considerable amount of salt in an average serving size .
if the meat used is of chicken and/or turkey , the content of polyunsaturated fatty acids is about a third of the nutritional requirement .
however , the quality of the meat used seemed to be low , when considering the collagen content .
the nutritional value of the dish seemed to be mainly influenced by the abundance of meat used in the preparation and less related to that of other components .
the consumption of an average portion of this rte food contributes with one - third and slightly less than half of the energy requirements for males and females respectively and it can be an alternative to one of the two daily meals , although with a moderate frequency of consumption . | the sampling activity for this study was performed between september and october 2012 .
it involved seven shops in verona , eleven in vicenza and two in its province ( bassano del grappa ) , northern italy .
the scope was to measure the values of energy and nutritional components and to identify the profile of fatty acids in a serving of ready to eat dner kebab .
the samples were collected according to the usual proportions of this preparation , keeping all the components ( bread , meat , vegetables and sauces ) separated in different bags . in the laboratory , each component was weighed and , after pooling , processed for the analytical determination of humidity , crude protein , lipid content and fatty acid profile , ashes , sodium ( salt ) , carbohydrate , collagen ( measured only in meat ) and fibre .
the results showed a highly standardized recipe , while the comparison between the two towns showed a significant difference in carbohydrate concentration ( mainly due to the quantity of bread used ) . by observing data on the serving sizes sampled ( 274 to 618 g ) and
the nutritional values obtained , dner kebab can be seen as a ready to eat dish providing much energy : on average a serving size covers 45 and 36% of the recommended daily intake of energy , 95.7 and 82.1% of protein , 42.5 and 33.4% of saturated fatty acids for females and males , respectively , and 85.5% of salt regardless of gender .
dner kebab can be considered as an occasional substitute to one of the two main meals of the day . |
although the incidences of gunshot wounds to the face[13 ] as well as penetrating spinal injuries[47 ] have increased during the past decades , craniofacial injuries caused by missiles are not generally as common as they are in other areas.[810 ] the association of gunshot traumas to the face with cervical spine injuries is infrequent : the reported incidence varies up to 8.1% of facial gunshot wounds . according to the study of kihtir et al .
, gunshots to the mid - face and orbit carry the highest risk for concomitant cervical spine injury ( up to 20% ) . there is consensus about the four main steps in the management of patients with gunshot wounds to the face : securing airway , controlling hemorrhage , identifying other injuries , and repairing the traumatic facial deformities .
however , the literature reports are controversial in terms of time and methods of subsequent surgical facial reconstruction .
a 24-year - old male patient , who was referred to the department of oral and maxillofacial surgery for evaluation of a left facial swelling , gave a history of the gunshot injury to his face .
the patient was conscious , walking , anxious , complaining of facial pain with a wound on the left side of the face .
the patient stated that the firearm used was a handgun at a close distance of around 2.5 m. physical examination revealed an entry wound in the left maxillary region , with subconjunctival ecchymosis but no exit wound [ figure 1 ] .
the patient was in good general condition without any signs of respiratory distress , hemorrhage , significant hematoma , retropharyngeal edema , or neural impairment .
radiography of the skull in the posterior - anterior ( pa ) projection [ figure 2 ] and lateral radiography of the cervical spine [ figure 3 ] revealed location of the foreign body at the level of c2 and c3 . there were no radiographic signs of damage to the surrounding bone elements . a computed tomography ( ct ) scan confirmed the presence of an isolated missile in relation to the cervical vertebrae [ figure 4 ] .
the injury was managed with conservative debridement and , after a series of wound dressing changes , 4 weeks of permanent monitoring , and antibiotic therapy , the patient was discharged from the hospital as there were no signs of infection or neurological changes .
the patient underwent regular follow - up for one year and no neurological complications were observed .
photograph of the patient showing a wound on the left maxillary region - the site of entry of the missile .
radiograph of the cervical spine in lateral projection showing the missile at the level of c2 and c3 vertebrae .
axial computed tomography section of the neck indicating position of the missile on the lateral aspect of the cervical vertebrae and fluid in the left maxillary sinus .
penetrating cervical spine traumas usually lead to spinal cord injury by direct spinal cord transection , contusion or ischemia due to arterial injury .
the main reason for not performing surgical bullet removal in the reported case was the absence of neurological deficit .
saxon et al illustrated possible delay in onset of clinical manifestation of cord injuries ( brown - sequard syndrome ) following a gunshot wound to the face . delayed complications due to migration of the impacted bullet are also described , although it was not always associated with neurological deficit .
the extent of tissue damage in gunshot wounds depends on the distance from which the gun is fired , missile track , and bullet structure , size , and velocity . in the reported case ,
a small - caliber missile of comparatively low velocity caused the injury by direct tissue crushing and laceration , producing the cavity that is not as large as can be seen in high - speed bullets , such as in rifle injuries .
the missile , track , passed through the left maxillary bone , ethmoidal labyrinth , and pharynx , and stopped in the left lateral to c2 and c3 vertebrae .
there are very few reported cases of transpharyngeal cervical spine injury , but most agree that progression of neurological deficit is an indication for urgent bullet removal.[1517 ] retained bullets rarely cause problems of delayed infection and late neurological decline , and only if a neurological deficit develops , which is possible after many years , should surgical intervention be considered . even in patients with static neural deficit ,
surgical decompression and bullet removal are sometimes not useful because of the absence of a significant effect on the neurological outcome and possible post - operative complications . in a reported case of fractures of the c1 anterior ring and the odontoid process ,
both associated with multiple bullet fragments , the patient 's motor and functional recovery supported the decision of not performing spinal surgery .
the clinical benefits of bullet removal to avoid lead toxicity or neurotoxic effects of copper are still unclear .
our report illustrates that knowledge of the path of the missile and close clinical observation of the patient are critical for assessment of management of patient with gunshot wounds to the face .
no surgery was performed , and the patient remained stable for a year without any complication or migration of the missile , which was lodged on the lateral aspect of c2 and c3 vertebrae . | an unusual case of facial gunshot injury with the missile lodged in the cervical spine region , but without any neurological impairment , is reported .
the extent of tissue damage and missile track termination in a male patient who sustained gunshot trauma to the face was assessed by plain radiography and by computed tomography scans .
the patient was treated conservatively and observed for clinical manifestations of neurological deficit for one year .
we present a case of gunshot injury to the face with the missile lodged in the cervical spine region and atypical absence of clinical manifestation that may occur even when a bullet remains in the vicinity of the cervical spine . |
hepatic abscess is an uncommon disease condition that has the potential for high mortality when untreated .
open surgical drainage was the usual treatment modality until the 1980s when image - guided percutaneous aspiration or drainage in addition to systemic antimicrobial agents became the standard of care .
outcomes in patients with this disease have therefore improved with the ready access to interventional radiology in its diagnosis and management .
indications for surgical drainage have now been limited to cases of failure of percutaneous drainage , complications of percutaneous drainage and presence of concomitant intraabdominal surgical pathology .
the dearth of trained radiologists and drainage facilities may still account for high rate of case referrals for surgical drainage especially in developing countries .
the laparoscopic approach to drainage offers an option that can reduce the increased morbidity and mortality associated with open surgery .
this study aims to evaluate the contribution of laparoscopy - guided to drainage of liver abscess in a public hospital in a developing country .
from september 2007 to december 2012 , 11 patients were referred to the surgery department of the federal medical centre , owerri from the internal medicine units . of these , eight patients were worked up for laparoscopic abscess drainage .
the case notes of these eight patients were reviewed and the following data were extracted : patient demographics , initial volume of fluid drained from the liver , duration of the operation , duration of hospital stay , and complications of the procedure .
complete blood count , liver function tests and coagulation assays were routinely done on the patients .
abdominal ultrasound scan was done in all patients , which indicated liver abscess ; one patient had a computed tomography ( ct ) scan in addition .
preoperative assessment showed abscess limited to the right lobe of the liver in five patients , multiloculated abscesses involving both lobes in three patients . under general anesthesia ,
the adhesions between the liver and the diaphragm as well as the anterior abdominal wall were freed to expose the area of the liver where the abscess was pointing .
aspiration of the cavity was done with veress needle passed percutaneously or with suction nozzle ; and in the process samples for bacterial cultures were taken .
finding of anchovy - sauce - like fluid supported the diagnosis of amoebic liver abscess .
thereafter , de - roofing of the cavity was done with electrocautery to enable insertion of a drainage catheter [ figure 1 ] .
from september 2007 to december 2012 , 11 patients were referred to the surgery department of the federal medical centre , owerri from the internal medicine units . of these , eight patients were worked up for laparoscopic abscess drainage .
the case notes of these eight patients were reviewed and the following data were extracted : patient demographics , initial volume of fluid drained from the liver , duration of the operation , duration of hospital stay , and complications of the procedure .
complete blood count , liver function tests and coagulation assays were routinely done on the patients .
abdominal ultrasound scan was done in all patients , which indicated liver abscess ; one patient had a computed tomography ( ct ) scan in addition .
preoperative assessment showed abscess limited to the right lobe of the liver in five patients , multiloculated abscesses involving both lobes in three patients .
under general anesthesia , initial peritoneal access for pneumoperitoneum was achieved usually with veress needle through the umbilicus .
the adhesions between the liver and the diaphragm as well as the anterior abdominal wall were freed to expose the area of the liver where the abscess was pointing .
aspiration of the cavity was done with veress needle passed percutaneously or with suction nozzle ; and in the process samples for bacterial cultures were taken .
finding of anchovy - sauce - like fluid supported the diagnosis of amoebic liver abscess .
thereafter , de - roofing of the cavity was done with electrocautery to enable insertion of a drainage catheter [ figure 1 ] .
eight patients were worked up with the intent of carrying out laparoscopic drainage of liver abscess .
they were receiving treatment in the internal medicine units for between 2 and 13 weeks before referral .
the ultrasound scan suggested the abscess cavity to be between 7 and 25 cm in diameter .
out of these , intraoperatively , one case of hemangioma and one case of multiple hepatic nodules were seen [ table 1 ] . among those that had confirmed liver abscess , the age range of patients was 31 - 54 years and male to female ratio
postprocedure microbiological analysis of aspirate yielded a positive culture of escherichia coli in one patient while cytology demonstrated acid fast bacilli in another .
this patient was monitored with serial ultrasound scan , at 6 weeks postprocedure the residual abscess cavity was 45 mm in diameter .
a paradigm shift did occur in the management of hepatic abscesses about four decades ago , and with it the exceptionally high mortality associated with this disease condition has been drastically reduced .
the first line of management has largely become ultrasound or ct - guided drainage in combination with antimicrobial agents .
amoebic liver abscess , a disease more prevalent in the tropics and low socioeconomic groups , is primarily treated with medications .
this approach has shown significant advantage in being safe and efficacious and low - cost .
failure of conservative therapy , rupture of the abscess , presence of multiloculated abscesses , and complications from percutaneous drainage may necessitate surgical intervention .
the surgical option also has the added advantage of accurate positioning of drainage catheter and simultaneous treatment of the abscess and underlying abdominal pathology .
various reports have explored the laparoscopic approach in the management of liver abscesses . whereas it could be argued that this may be an unnecessarily expensive modality of treatment , the known benefits of minimally invasive surgery present a compelling response .
it helps in the early return of gastrointestinal function and resumption of normal activities ; on the other hand it potentially achieves a better abscess drainage .
there was neither mortality nor conversion to open surgery in our series , which is in line with other reports in the literature .
the value of a trained radiologist can not be discounted for the successful conduct of percutaneous drainage .
trained personnel may be a challenge in some developing countries , a reason for continued persistence with open surgical drainage .
the case of liver hemangioma and carcinoma in our series should not have been misdiagnosed preoperatively .
utilization of laparoscopic ultrasonography may be another added advantage compared with transabdominal in localizing the abscess site for drainage .
a small number of cases and the retrospective design are limitations of the present study .
however , we seek to highlight the possible value of laparoscopy in improving diagnostic and therapeutic capacities in our peculiar environment . we also want to demonstrate the modest progress laparoscopic surgery is making from its humble beginnings in our sub - region . | background : image - guided percutaneous drainage with antimicrobial agents is the standard modality of treatment of liver abscess .
open surgical drainage , and lately laparoscopic drainage becomes useful in selected patients .
nigeria is awakening late to the laparoscopic surgery revolution .
public health institutions have started making enormous investments in minimal access surgery , which can augment deficient diagnostic capacities.objective:to describe the outcomes of the patients who underwent laparoscopic liver abscess drainage at the federal medical centre , owerri.materials and methods : a retrospective analysis of the laparoscopic liver abscess drainage procedures done between the period september 2007 and december 2012 was done.results:a total of eight patients in the study period were worked up for abscess surgical drainage based on ultrasound ( seven cases ) and computed tomography ( one case ) supported localized collection in the liver .
intraoperatively , one patient was noticed to have nodules on the liver that was later confirmed as hepatocellular carcinoma .
operating time ranged from 37 to 126 min .
there was no conversion to open surgery . on the follow - up ,
one patient had residual abscess of 45 mm diameter size , after 6 weeks , and in whose aspirate acid - fast bacilli were identified.conclusion:laparoscopic drainage should be considered in the management of liver abscess . |
loffler s syndrome ( simple pulmonary eosinophilia ) are characterized by minimal pulmonary symptoms , transient pulmonary infiltrates that tend to be patchy , and mild peripheral blood eosinophilia .
although clonorchiasis is a common hepatobiliary disease in korea that produces marked eosinophilia in the peripheral circulation , its clinical presentation rarely includes pulmonary infiltrates . in the medline search of english language articles published from 1960 through 2003 , only two cases of eosinophilic pulmonary infiltrations associated with clonorchis sinensis were found .
we report a case of loffler s syndrome associated with clonorchis sinensis infestation in korea .
a 54-year - old man presented with a several - day history of erythematous rashes on his extremities .
he also had a history of ingesting raw freshwater fish , but not of bronchial asthma or other medical side effects .
he had visited a community clinic where a chest radiograph showed a solitary pulmonary nodule in the right upper lobe ( figure 1 ) .
the rashes subsided following treatment with an antihistamine and a non - steroidal anti - infilammatory agent .
the patient s physical examination was unremarkable . on admission , his leukocyte count was 11,250 cells/l with 38% neutrophils , 23% lymphocytes , 4% monocytes , and 35% eosinophils .
other hematologic parameters were 13.5 g / dl hemoglobin , 7 mm / hr esr , 12.5 sec ( control=11.5 sec ) pt , and 28 sec ( inr=1.0 ) ptt .
anti - hepatitis b surface antigen was negative , but anti - hepatitis b surface antibody was positive .
stool examination for helminths revealed many clonorchis sinensis ova ; stool ova count was 200 eggs per gram .
a skin test for paragonimus westermani was negative , but one for clonorchis sinensis was strongly positive .
total serum ige was 1020 iu / ml and clonorchis - specific igg antibody by micro - elisa was 0.27 ( < 0.25 ) .
serial chest radiography ( figure 2a , 2b ) and high resolution computed tomography ( figure 3a , 3b , 3c ) showed that the pulmonary nodules and patchy densities migrated to other sites and changed in shape and size daily . on the second day of admission , the patient underwent fiberoptic bronchoscopy that revealed no endobronchial lesion .
the bronchoalveolar lavage ( bal ) fluid had cell proportions of 45% macrophage 15% lymphocytes , 15% neutrophils , and 25% eosinophils ( figure 4 ) .
the bal fluid was negative for bacterial , acid - fast bacilli , and fungi .
abdominal ultrasound revealed no specific abnormalities except mild splenomegaly . on the ninth day of admission , migrating nodular densities on chest radiographs decreased and then disappeared dramatically following oral praziquantel therapy ( figure 6 ) .
in 1932 , loffler described a syndrome of self - limiting , transient pulmonary infiltrates associated with blood eosinophilia and mild pulmonary symptoms such as cough , malaise , fever , and constitutional symptoms .
loffler described the associated radiographs as showing migratory shadows that were sometimes homogeneous , but more often spotty or cloudy , of variable delineation ( single or multiple , unilateral or bilateral ) that disappeared in three to eight days .
radiography of this syndrome typically shows migrating bilateral infiltrates of a non - segmental distribution and fluffy alveolar infiltrates in the upper lobes .
eosinophilic lung diseases have been given variable names on the basis of etiology , clinical manifestation , and their pathophysiology .
for example , crofton et al . divided them into five catergories , and reeder and goodrich used the term pulmonary infiltration with eosinophilia ( pie ) syndrome . in 1969 ,
liebow and carrington defined pulmonary infiltrations of the lung by eosinophils , which may or may not be accompanied by an excess of eosinophils in the peripheral blood .
currently eosinophilic lung diseases are classified or defined by the severity of symptoms , abnormalities on chest radiography , and by the presence and extent of peripheral blood eosinophilia .
histopathological findings in these cases disclose dense aggregates of eosinophils , histiocytes , and multinuclear giant cells with alveolar spaces , interstitium , and bronchioles with scattered lymphoctes and plasma cells .
the pulmonary eosinophilias are a diverse group of disorders whose etiologies include various pulmonary infections ( especially parasites , aspergillus , and mycobacteria ) , some tumors ( hodgkin s lymphomas ) , drugs , chemicals , pollens , and collagen vascular disorders . at the time of presentation ,
most bacterial pneumonias , however , can be virtually ruled out by the presence of increased percentages of eosinophils .
eosinophilia may be noted in pneumocystis carinii pneumonia , fungal pneumonia , and parasitic infections involving the lung , such as strongyloides stercoralis , filaria , toxocara , ascaris , paragonimus , and schistosoma .
drugs reported to cause clinical presentations similar to loffler s syndrome include nitrofurantoin , phenytoin , l - tryptophan , acetaminophen , ampicillin , heroin , and cocaine .
clinical clues that favor drug - induced pulmonary eosinophilia the presence of rash or blood eosinophilia .
eosinophils often play an important role not only in host defense mechanisms ( due to their lethal effects against helminths , protozoa , fungi , and other microorganisms ) but also in various pulmonary infections , some tumors , and collagen vascular diseases . two mechanisms of pulmonary eosinophilic infiltrations in parasitic infestations have been postulated in the literature . the first is direct invasion of the lung as in ascaris , schistosomes , filaria , p. westermani , acylostoma duodenale , necator americanus , strongyloides , or echinococcus .
second , allergic reactions to entameba histolytica , t. trichiura , toxocar canis or clonorchis sinensis may be the cause . in cases such as this
one , immunologic stimulation from the life cycle of clonorchis sinensis in humans may cause the pulmonary infiltrations .
the eosinophilic chemotaxis in parasitic infestations may result from one or more of the following mechanisms : 1 ) ige - mediated reactivity against the parasite 2 ) a direct chemotactic property of certain parasites 3 ) t cell - dependent mechanisms and 4 ) immune complex mechanisms .
the pulmonary infiltrates themselves may represent a combination of migrating organisms as well as a pulmonary reaction against them . in our patient with migrating pulmonary infiltration
, the hypersensitivity response may be the major factor . when faced with a patient having signs consistent with eosinophilic pulmonary infiltrations , a differential diagnosis
should be included to exclude other disorders such as collagen vascular disease , allergic bronchopulmonary aspergillosis , bronchocentric granulomatosis , sarcoidosis , hodgkin s disease , hypereosinophilic syndrome , helminthic , fungal infection , and drugs .
infestation with clonorchis sinensis , or so - called liver fluke , is a very common disease in korea involving the liver and intestines .
significant amounts of eosinophilia and marked leukocytosis are present in most koreans infected by clonorchis sinensis . however , pulmonary infiltrations have been reported in only one case in korea . in that case , diffuse bilateral nodular pulmonary infiltrations were the initial chest x - ray finding .
but in our case , eosinophilic pneumonia appeared as a solitary pulmonary nodule , with multiple migrating nodules thereafter developing on the other sites .
we can not exactly explain why eosinophilic pulmonary infiltration associated with clonorchis sinensis is rarely reported despite the higher prevalence of hepatobiliary clonorchiasis in korea .
this could be due to an exceptionally low level of this sort of co - morbidity so low as to be very rarely detected .
alternatively , as the case reported here , the eosinophilic pneumonia due to clonorchis sinensis could be very transient with few or no symptom that it could elude an examining physician .
we suspect that this latter explanation is more likely to be accurate due to the transient , self - limiting , and relatively asymptomatic nature of the pulmonary involvement .
this case enlarges the growing list of etiologic agents in the production of the so - called loffler s syndrome . | in 1932 , loffler described a syndrome of self - limiting , transient pulmonary infiltrates associated with peripheral blood eosinophilia and mild pulmonary symptoms .
a number of conditions are related to pulmonary eosinophilia or pulmonary infiltration with eosinophilia .
especially , parasitic infestations are often related to pulmonary eosinophilia , but only two cases associated with clonorchis sinensis have been anecdotally reported in english literature . here
we report a case of migrating pulmonary eosniophilic infiltrations associated with clonorchis sinensis that was successfully treated with praziquantel .
clonorchiasis should be considered in patients with marked eosinophilia and pulmonary infiltrations . |
one health is a concept that promotes the integration of human , animal , and environmental health by increasing communication and collaboration across different disciplines .
advances in food safety were largely due to the efforts of rudolf virchow ( 18211902 ) , a german physician who established the field of veterinary pathology and public health meat inspection programs that transformed the role of veterinarians into vanguards of public health ( 8) .
cross - disciplinary efforts waned in the 20th century , as medicine and science became increasingly specialized and reductionistic in approaching health and disease .
however , growing human populations , diminishing biodiversity , emerging infectious diseases , intensive agriculture , food safety and security , deforestation , global trade and travel , climate change , and others demand that a new paradigm be used to address these issues : a one health paradigm .
master 's degree programs in public health typically focus in core competency areas of biostatistics , epidemiology , health policy and management , social and behavioral sciences , and environmental health .
however , in a global , increasingly interconnected world in which land degradation , deforestation , intensive agriculture , global food production , water contamination , energy production , loss of biodiversity , antimicrobial resistance , and climate change contribute to emerging diseases , a broad - based one health approach becomes increasingly critical for global health .
there is a great need for professionals with education and training that span human , animal , and environmental health .
the deadly e. coli 0104:h4 outbreak centered in northern germany during may and june 2011 has devastated parts of the european agricultural industry , sickened thousands , and killed 32 people so far , highlights the need for interdisciplinary collaboration ( 9 ) .
the source of this outbreak , which likely involved the contamination of food with bacteria , remains uncertain , but it is likely ( based on precedent ) that domestic livestock were the source of fecal contamination .
logically , veterinarians who understand animal husbandry would be engaged in the investigation of this outbreak .
currently , there are only 71 accredited ( by aavmc standards ) schools of veterinary medicine ( 10 ) , and the number of graduates pursuing careers in public health does not meet global needs .
this outbreak caused huge disruption of commerce and threatened the entire fabric of public health .
interestingly , it was a surprise to those responsible for public health that veterinarians were wrestling with related coronavirus diseases of domestic livestock and companion animals for many years , and that there was a depth of knowledge in the veterinary community that was applicable to sars .
schools of public health could play a tremendous role in bridging human , animal , and environmental health by offering master 's and doctoral degree programs in one health .
in addition to studying biostatistics and epidemiology , students would study food safety and security , ecosystem and environmental health , health issues of land degradation and urban development , agriculture and sustainability , health impact of water and energy usage , biodiversity and zoonotic diseases , among others .
solutions to global challenges will not be developed until a cadre of multi - disciplinary scientific professionals communicate and collaborate on work that integrates the intricate linkages between human , animal , and environmental health .
public health policy and practice must reflect this fact . preventing and confronting future outbreaks will require experts with education and training in human , animal , and environmental health .
schools of public health can fill this need by providing degree programs in one health .
| this commentary offers suggestions for improving public health and public health education by emphasizing one health principles , the integrating of human , veterinary , and environmental sciences .
one health is increasingly recognized as a powerful approach to the prevention and control of zoonotic diseases , increasing food productivity and safety , improving biosecurity , and enhancing many areas of biomedical research . |
fibromuscular dysplasia ( fmd ) is a rare disease affecting small and middle - sized arteries of the kidney and brain , predominantly in adult women .
it rarely occurs in children and has not previously been treated with thrombolytics following acute stroke .
a 12-year - old girl was admitted to the pediatric emergency room after having collapsed during a soccer game .
family members reported that she had fallen down with convulsions of all four extremities . on admission , a neurological examination revealed a severe left - hemispheric syndrome with global aphasia , conjugated gaze deviation to the left , severe hemiparesis with extensor plantar response and hemihypesthesia of the right side [ national institute of health stroke score ( nihss ) : 19 ] . both the family history and her medical history were unremarkable apart from a migraine without aura .
acute cerebral mri showed an acute middle cerebral artery ( mca ) stroke on diffusion - weighted images involving the insula , the lentiform nucleus , the head of the nucleus caudatus and the centrum semiovale ( fig .
1a ) , with occlusion of distal m3 branches of the left mca and irregular contrasting of both the proximal left m1 and the distal left internal carotid artery ( ica ) segments ( fig .
1b ) revealed an extended perfusion deficit beyond the territory of the diffusion - weighted image lesions in the cortical areas of the complete mca territory , characteristic of a significant mismatch between the hypoperfused area and the diffusion impairment .
the patient was treated within an extended time window of 5 h after onset of the symptoms with intravenous t - pa ( 0.9 mg / kg body weight , resulting in 5 mg as a bolus and 50 mg continuously over 60 min ) .
forty - eight hours later , marginal improvement was observed ( nihss after thrombolysis : 15 ) .
laboratory studies were unremarkable as were an extended coagulation profile , vasculitis screening and analysis of the cerebrospinal fluid .
transthoracic and transesophageal echocardiography showed no abnormalities , while both extracranial and transcranial duplex sonography demonstrated a severe stenosis in the siphon of the left ica with significantly decreased prestenotic flow velocity .
furthermore , the sonography results showed a retrograde flow direction in the left proximal anterior cerebral artery , indicating hemodynamic compensation from right to left anterior circulation .
the size of the ischemia on a follow - up mri the next day was unchanged and without secondary hemorrhagic transformation . on mr angiography ,
all 3 mca segments , including the distal m3 branches , were recanalized while mild pathology within the proximal m1 and the distal ica persisted .
digital subtraction angiography of these vessel segments showed typical constrictions with ' strings of beads ' characteristic of fmd ( fig .
another follow - up mri at day 16 showed a new though clinically asymptomatic
ischemic lesion in the left mca territory , which prompted oral anticoagulant treatment with phenprocoumon ( inr 2.0 - 3.0 ) .
the patient was released into rehabilitation after further improvements ( nihss on discharge : 8) and presented with severe paresis of the right arm and moderate paresis of the right leg .
three months later we saw her again with further clinical improvement ( nihss : 5 ) : only moderate right spastic hemiparesis and mild residual aphasia persisted .
stroke in children is rare , with an incidence varying from 1.3 to 13.0 per 100,000 .
while systemic thrombolysis with t - pa is considered the gold standard of acute treatment in adult ischemic stroke patients [ 2 , 3 ] , insufficient data on systemic intravenous or local intra - arterial thrombolysis in children are available [ 4 , 5 , 6 ] . a major problem is the delay in diagnosis of stroke , which is even more challenging than in adults , as this diagnosis is rarely assumed by parents , family members or doctors .
although stroke in young adults is often due to similar causes as those in the elderly , active mechanisms in children are different .
fmd is very rare in children and has only been reported in a few casual communications [ 8 , 9 , 10 ] . as mr angiography is unable to provide sufficient resolution to demonstrate small multisegmental structural changes of commonly multiple brain - supplying arteries , digital subtraction angiography is needed . therefore , fmd of the carotid or intracranial arteries often remains undiagnosed .
the use of platelet inhibitors or oral anticoagulation for 6 - 12 months after clinical events in adults is a possible regimen in children as well . as a differential diagnosis , transient cerebral angiopathy ( tca )
was discussed . despite being frequently considered as a reason for stroke in children with focal stenosis of the intracranial vessels , the diagnosis of tca
does not describe a particular underlying pathophysiology and , other than an association with varicella zoster infections , there is little known about its etiology .
an overlap of tca with fmd can not be excluded . to our knowledge , this is the first report of a child with fmd treated with t - pa , and also the first one that describes thrombolysis of an acute stroke which was based on mri diffusion / perfusion mismatch criteria , in a child . | fibromuscular dysplasia , predominantly found in adult women , is a rare disease of small and middle - sized arteries of the kidney and brain .
we present a case of a 12-year - old girl with acute ischemic stroke , due to fibromuscular dysplasia of the distal internal carotid artery and the proximal middle cerebral artery , which was successfully treated with t - pa . |
cisplatin or cis - diamminedichloroplatinum ( ii ) ( cddp ) is an anticancer drug for treatment of solid tumors [ 1 , 2 ] .
one of the major side effects of cddp is nephrotoxicity , which is known as a limitation for cddp therapy in clinic .
inflammation , oxidative stress , and change in the renal circulation may also be induced by cddp [ 46 ] . to decline cddp - induced nephrotoxicity ,
losartan is an angiotensin type-1 receptor blocker as well as an antioxidant , which has been suggested to be nephroprotective against cddp - induced nephrotoxicity by others [ 10 , 11 ] .
losartan may reduce renal disease progression in patients with type 2 diabetes and decrease hypertension and microalbuminuria or proteinuria [ 14 , 15 ] .
several studies have presented different effects of losartan on nephrotoxicity induced by nephrotoxic compounds such as cddp [ 10 , 11 , 16 , 17 ] .
losartan may prevent nephrotoxicity caused by administration of single dose of cddp in males [ 11 , 16 , 17 ] , but it promotes renal damage in females . although the nephroprotective role of losartan against single dose of cddp was reported in studies , cddp is clinically administered sequentially .
therefore , we designed the current study to investigate the effect of losartan on renal toxicity induced by sequential ( daily ) infusion of cddp .
forty - two male wistar rats ( animal centre , isfahan university of medical sciences ) weighing 175.56 2.24 g were used .
this research was approved in advance by the isfahan university of medical sciences ethics committee .
groups 1 and 2 , as the negative control groups , received intraperitoneal ( ip ) losartan 10 and 20 mg / kg / day for a period of 10 days , respectively .
group 3 , as the positive control group , received saline for a period of 10 days , but from day 3 the animals received cddp ( 2.5 mg / kg / day , ip ) for the next seven days .
groups 4 and 5 received treatment regimen the same as groups 1 and 2 , respectively , but from day 3 they received cddp for the next seven days .
the animals ' body weight was recorded on a daily basis . at the end of the experiment ,
all animals were anesthetized by ketamine ( 75 mg / kg ) to obtain blood samples via heart puncture .
the left kidney was fixed in formalin for pathological investigations , and the right kidney was homogenized and centrifuged at 15000 g and the supernatant was kept in 20 until measurement .
the levels of serum creatinine ( cr ) , blood urea nitrogen ( bun ) , total proteins ( tp ) , magnesium ( mg ) , and lactate dehydrogenase ( ldh ) were measured using diagnostic kits ( pars azmoon , tehran , iran ) .
the serum and kidney levels of nitrite ( stable nitric oxide metabolite ) were measured using eliza kit ( promega corporation , madison , wi , usa ) .
the serum and kidney levels of malondialdehyde ( mda ) were measured by a manual method .
briefly , 0.5 cc of the sample was added to 1 cc of trichloroacetic acid 10% .
, 0.5 cc of the supernatant was mixed with 0.5 cc of thiobarbituric acid 0.67% and placed in the boiling water for 10 minutes . after cooling , the absorbance was determined at the wavelength of 532 nm .
the left kidney was fixed in neutral formalin 10% and embedded in paraffin . after slicing , hematoxylin and eosin staining
was performed to examine the samples for tubular atrophy , as well as the presence of casts , debris , and necrotic materials in the tubular lumen .
intensity of the tubular lesion was scored from 1 to 4 , while zero was assigned to the normal tissue without damage .
the levels of bun , cr , ldh , mda , tp , mg , nitrite , and body and kidney weights were analyzed by the one - way anova followed by the lsd test .
tissue damage scores were compared by the kruskal - wallis or mann - whitney tests .
the results revealed that nephrotoxicity was induced by cddp , which was characterized by significant increase in the serum levels of bun and cr ( p < 0.05 ) .
administration of losartan at the doses of 10 and 20 mg / kg did not alter the serum levels of bun and cr , while coadministration of cddp and losartan elevated the serum levels ( 10 mg / kg , insignificantly , and 20 mg / kg , significantly , p < 0.05 ) , when compared with the cddp alone ( positive control ) group ( figures 1 and 2 ) .
cddp significantly elevated the serum level of mda ( p < 0.05 ) when compared with the negative control groups .
however , administration of losartan ( 10 and 20 mg / kg ) accompanied with cddp attenuated the serum level of mda when compared with the cddp alone treated group ( positive control group ) ( p < 0.05 ) . considering kidney tissue mda level ,
no significant difference was observed between the losartan alone - treated groups ( 10 and 20 mg / kg ) and the cddp alone treated group ( p < 0.05 ) .
however , coadministration of losartan ( 20 mg / kg ) and cddp significantly increased the kidney tissue level of mda ( p < 0.05 ) when compared with the cddp alone treated group ( figures 1 and 2 ) .
the serum level of nitrite in the positive control group significantly increased compared with the negative control groups ( p < 0.05 ) , while coadministration of cddp and losartan ( 10 and 20 mg / kg ) decreased the serum level of nitrite insignificantly .
the kidney tissue level of nitrite was decreased by cddp alone , and combination of losartan ( either 10 mg / kg or 20 mg / kg ) and cddp did not alter the results ( figures 1 and 2 ) .
tissue level of ldh in the positive control group was significantly decreased when compared with the negative control groups ( p < 0.05 ) . however , coadministration of losartan ( 10 and 20 mg / kg ) and cddp compared to administration of cddp alone increased the tissue level of ldh insignificantly ( figures 1 and 2 ) . no significant difference in the tp serum level
the serum level of mg was significantly increased by cddp plus losartan ( 20 mg / kg ) when compared with the positive control group ( p < 0.05 , figures 1 and 2 ) . cddp reduced bodyweight .
administration of 20 mg / kg losartan accompanied with cddp significantly ameliorated weight loss ( p < 0.05 ) .
cddp also increased kidney weight significantly ( p < 0.05 ) , while coadministration of losartan ( either 10 mg / kg or 20 mg / kg ) and cddp did not alter the results ( figures 1 and 2 ) .
cddp induced kidney damage when compared with the negative control groups ( p < 0.05 figures 1 and 2 ) .
in addition , cddp accompanied with losartan ( 10 and 20 mg / kg ) intensified the damage .
high dose of losartan plus cddp provided significant increase in kidney tissue damage when compared with the cddp alone treated group ( p < 0.05 ) ( figures 2 and 3 ) .
in the current study , we attempted to investigate the effect of losartan on renal toxicity induced by daily administration of cddp .
we found that losartan is not a nephroprotectant agent against cddp nephrotoxicity in this rat model .
cddp affects kidney and body weight ; serum cr , bun , tp , osmolality , and nitric oxide levels [ 18 , 19 ] and previous reports indicated that losartan is a nephroprotectant agent against cddp - induced nephrotoxicity [ 10 , 11 , 16 ] .
saleh et al . reported administration of single dose of cddp and losartan reduced kidney toxicity . in our pervious studies
, we also administered single dose of cddp and found that losartan is nephroprotectant in males [ 10 , 11 ] but not in females [ 11 , 20 ] .
in contrast , deegan et al . reported that injection of low and high single doses of losartan ( 10 and 30 mg / kg ) two hours prior to cddp injection could not ameliorate nephrotoxicity induced by cddp .
reported that losartan intensifies nephrotoxicity induced by gentamicin , while coadministration of cimetidine ( as a cytochrome - p450 inhibitor ) and losartan could decrease the nephrotoxicity induced .
losartan also has inconsistent effects on renal function and induces renal vasodilation , inhibits reduction of glomerular filtration rate in hypertension , decreases proteinuria , and reduces morbidity and mortality in diabetic nephropathy [ 23 , 24 ] .
as mentioned above , we observed that administration of losartan promotes renal dysfunction induced by cddp , which is shown by increasing the values of bun , cr , and kidney tissue damage score .
in addition , it seems that continuous or single dose administration of cddp may induce different levels of kidney tissue damage . in the present study ,
the animals received cddp in total amount of 17.5 mg / kg ( more than twice of single dose of cddp in our previous study ) .
therefore , the cumulative dose of cddp increased in the kidneys and enhanced renal toxicity .
this finding is in agreement with those reported in other studies [ 26 , 27 ] .
high dose of losartan ( 20 mg / kg ) ameliorated the bodyweight loss .
losartan affects aldosterone secretion , and this may lead to retention of water and electrolytes to prevent the weight loss induced by cddp .
kidney weight was also increased by cddp that could be related to retention of water and solutes in renal tissues or changes induced by cddp in renal blood flow .
our results showed that administration of cddp increased serum level of mda as a final product of lipid peroxidation and administration of losartan ( 10 and 20 mg / kg ) as an antioxidant could ameliorate this .
it seems that losartan can reduce the production of oxygen free radicals and inhibit the oxidative pathway .
an evidence showed that telmisartan , an angiotensin ii receptor antagonist ( angiotensin receptor blocker , arb ) , can inhibit the production of oxygen free radicals and improve renal function . in the current study , it was also observed that cddp increased the serum level of nitrite and decreased kidney tissue level of nitrite .
inos can produce no in the body more than enos and nnos [ 3133 ] .
our results showed that administration of cddp alone did not change the serum levels of mg and tp .
it is documented that serum level of mg decreased in the third week after cddp administration .
proximal tubule is the main site of protein reabsorption and cddp degenerates this structure and subsequently protein uptake is reduced and protein urinary excretion is increased .
therefore , our observations associated with mg and tp may be related to the experiment protocol .
the supportive role of losartan in preventing cardiovascular disease or even reducing nephrotoxicity induced by cddp [ 10 , 11 , 16 ] has been reported previously . however , in the current study , it was found that losartan could not improve renal toxicity induced by continuous administration of cddp and also intensified renal failure .
carrying out further investigations is necessary to investigate the combination of losartan and other agents . | background . cisplatin ( cddp ) is an anticancer drug , which is accompanied with major side effects including nephrotoxicity .
we tested two doses of losartan ( 10 and 20 mg / kg / day ) against nephrotoxicity in a rat model treated with daily administration of cddp ( 2.5 mg / kg / day ) . methods .
five groups of rats were examined .
groups 1 and 2 received losartan 10 and 20 mg / kg / day , i.p , for a period of 10 days .
group 3 received saline for 10 days , but from day 3 the animals received cddp ( 2.5 mg / kg / day , i.p ) for the next seven days .
groups 4 and 5 received treatment regimen the same as groups 1 and 2 , but from day 3 they also received cddp for the next seven days . at the end of the experiment ,
blood samples were obtained and the kidneys were removed to undergo pathological investigation and to obtain supernatant from homogenized tissue . results .
cddp induced nephrotoxicity , but the serum levels of creatinine and blood urea nitrogen were not attenuated by losartan .
the pathological findings confirmed that losartan did not have nephroprotective effect in this experimental model .
conclusion . according to the findings
, losartan could not improve renal function impaired by toxicity induced by continuous doses of cddp , and also it worsened the renal failure . |
falls among the elderly constitute a significant health problem , as approximately every
third person over 65 and every second individual over 85 falls at least once a year1 , 2 .
the multi - disease phenomenon may significantly negatively affect functional dexterity in the
elderly and result in gait and balance impairment .
various consequences of somatic diseases ,
combined with reduced dexterity and physical activity , influence the quality of life in
older adults , especially those who have already lost independence for activities of daily
living ( adl ) , and are residents of nursing homes .
their independence is usually lower and
they belong to the group at high risk of falling .
thus , fall risk assessment should include
various tests for static and dynamic balance .
the physical and functional consequences of falls ( fractures , soft tissue injury , premature
institutionalization , and death ) have been well documented .
long - term consequences may
include loss of independence , restriction of physical activity , lowered quality of life ,
increased social isolation , or depression3 , 4 .
fear of falling affects psychological
well - being and constitutes an independent risk factor for reduced mobility and lowered
quality of life5 .
numerous methods have been developed to evaluate balance and risk of falling among older
adults , including the timed up and go ( tug ) test , tinetti performance oriented mobility
assessment ( poma ) test , berg balance test ( bbs ) , and one - legged stance test ( olst ) .
these
tests assess various parameters , especially standing balance , stepping ability , general
function , reaction time , lower limb strength , dual tasking , gait variability , gait cadence ,
and vision ( visual acuity , contrast , and field)6 .
they may also identify those at risk of falling who require
lifestyle interventions , including exercises and home environment safety modifications7 , 8 .
a
physiotherapist , as a member of the geriatric team , should implement adequate preventive and
therapeutic measures .
the aim of the study was to determine which gait and balance tests generated the most
reliable results for assessment of risk of falling , based on the number of falls over the
last 12 months .
the study was conducted in accordance with the
declaration of helsinki and the local ethics committee approved the study protocol ( no .
this study was conducted in 5 nursing homes in poznan , poland . out of 400
individuals ( aged > 65 )
, a group of 153 residents who were able to walk a distance of 3 m
unaided or using orthopedic equipment ( i.e. , performed the tug test ) and scored > 18
points on the mini - mental sate examination ( mmse ) were identified .
patient characteristics
are presented in table 1table 1.patient characteristicscharacteristicsmalefemalenumber of patients 31122age ( years)mean sd73.0 8.077.6 8.1median ( range)72 ( 6581)77 ( 6594)educationprimary school ( completed 7 years)639vocational school ( completed 3 years)1414high school ( completed 4 years)755university degree ( completed 5 years)414number of diseasesdegenerative disease1476stroke411hypertension1659osteoporosis27diabetes mellitus 517ischemic heart disease 646visually impaired316others1586number of medicationsmean sd3.2 3.42.7 3.7median ( range)1 ( 09)1 ( 014)barthel scale ( points)mean sd96.2 6.489.8 13.2median ( range)100 ( 85100)95 ( 45100)mmse score ( points)mean sd25.4 2.826.5 3.1median ( range)25.5 ( 1930)27
( 1930)number of falls over the last 12 monthsmean sd1.03 1.61.01 1.7median ( range)0 ( 06)0 ( 08 ) .
comprehensive geriatric assessment was performed in 153 subjects to determine
the program of rehabilitation .
various scales were used , including the barthel scale to
measure adl performance , ( 020 points : very dependent , 2185 : moderately dependent , 86100 :
independent)9 .
the mmse was used to
evaluate cognitive function10 , which in
older adults aids in design of the physiotherapy treatment .
the scale assesses 5 areas of
cognitive function : orientation , registration , attention and calculation , recall , and
language .
tests to predict the risk of falling have been used to evaluate balance in the elderly ; for
example , tug assesses proactive balance .
the patient is instructed to rise from the chair
( approximate seat height of 46 cm ) , walk at a comfortable and safe pace to a line on the
floor 3 m away , then turn and walk back to the chair and sit down again .
patients should wear their usual
footwear and may use their customary walking aid ( cane or walker ) , if necessary11 .
a score of < 10 s is normal , 1120 s
signifies subjects with frailty syndrome or slight disability , and a score of > 20 s
indicates that the individual requires a significant amount of help from others while
walking , and usually heralds the need to implement an intervention . a tug score of 13.5
poma consists of 2 parts for assessment of balance and gait , and
is frequently used to evaluate balance in elderly populations .
scores of 2619 reveal the presence of a problem , which might
lead to a fall , whereas a score of 2628 is normal12 .
it consists of 14
items , with a maximum total score of 56 points .
bbs scores have been identified for the ability to walk without an
aid ( 49/56 points ) and the ability to walk without a 4-wheeled walker ( 43/56 points)14 .
steady - state balance may also be
evaluated with the use of olst , which is used as a screening method to determine balance
impairment in elderly populations . the person is instructed to stand on one leg without
support of the upper extremities .
the maximum test time is 30 s. in this study , the participants were instructed
to keep their eyes open .
normal ranges with the eyes open are : 6069 years : 22.5 8.6 s ,
and 7079 years : 14.2 9.3 s15 .
gehlsen
and whaley demonstrated that people who fall usually score lower on this test , whether with
the eyes open or closed ( 10.9 s vs. 18.7 s , p<0.001 , and 3.6 s vs. 5.2 s , p<0.05)16 . in this study ,
the results were analyzed
according to the number of falls during the last 12 months .
quantitative variables are presented as mean and standard deviation . due to the
non - parametric distribution of some variables , median and range of parameters
were taken
into account . the mann - whitney test , spearman s rank correlation coefficient , and the deming
linear regression ( because parameters were in different units : points and seconds ) were used
in the analysis
the results of the tests are presented in table
2table 2.gait and balance assessmentparametersresultsfallersnon- fallersage ( years)mean76.67 8.3 78.7 7.975.2 8.3 * median 76.57974range6594 6594 6591 tug ( sec)mean18.6 9.3 20.75 9.2 16.5 9.1 * median 161614range552 548052poma ( pts)mean20.4 5.7 19.1 5.7 21.4 5.4 * median 222023range428428828bbs ( pts)mean41.3 12.738.9 13.443.1 11.9 * * median 454446range056056456olst ( sec)mean5.3 7.83.5 6.06.6 8.6 * * median 204range030027s030sn=71 , * p<0.01 , * * p<0.05 . out of 153 subjects ,
only 46% were deemed eligible for the olst test . deming
linear regression ( n=153 ) revealed statistically significant correlations between the number
of falls , longer results for tug r=0.27 ( p<0.001 ) , and lower scores for poma r=0.24
( p<0.001 ) and olst r=0.17 ( p<0.01 ) .
table
3table 3.correlation between number of falls in males and females , age and tests
results genderage ( years)tug ( sec)poma ( pts)bbs ( pts)olst ( sec)male ( n=31)r=0.5046*r=0.5627**r=0.3739***r=0.3450r=0.4027***female ( n=122)r=0.1811***r=0.2393*r=0.2294***r=0.1380r=0.1314*p<0.01 , * * p<0.001 , * * * p<0.05 presents results after the study group was subdivided according to gender .
a
statistically significant correlation between age and the number of falls in males
( p<0.01 ) and females ( p<0.05 ) was detected with spearman s correlation .
a high
correlation was also found between the number of falls and both longer tug results ( males
p<0.001 , females p<0.01 ) and lower poma scores ( males p<0.05 , females p<0.05 ) .
no correlation was found between gender and the bbs score , whereas results of the olst test
correlated with gender only in the case of males ( p<0.05 ) .
n=71 , * p<0.01 , * * p<0.05 * p<0.01 , * * p<0.001 , * * * p<0.05
based on the results , the evaluation of balance in fallers and non - fallers among elderly
residents of nursing homes showed that all tests , i.e. , tug , poma , bbs , and olst , enabled
adequate assessment of balance and identification of patients in need of interventions to
prevent falls . in nursing homes , it is essential to apply tests that will identify people
who need services to prevent falls .
the olst was least useful , as it could only be conducted
in 46% of the subjects .
the difference between fallers and non - fallers was statistically
significant in a comparison of tug ( p<0.01 ) and poma ( p<0.01 ) results . for bbs and
olst ,
a difference was present at a level of significance of p<0.05 . compared with
non - fallers , tug ,
poma , bbs , and olst scores in fallers were 4.25 s longer , 2.3 points
lower , 4.2 points lower , and 3.1 s shorter , respectively .
also evaluated
balance in fallers vs. non - fallers , but used bbs and computerized dynamic posturography
( cdp ) .
the bbs and cdp results in fallers were significantly lower compared to
non - fallers17 .
this study confirmed that the risk of falling increases with age in both males and females
over 65 .
nakano et al . demonstrated that female patients in their 70s show a significant
decline in functionality , i.e. , physical performance and balance , as compared to their male
peers .
older females display greater loss of muscle mass and muscle strength , which
predisposes to frailty syndrome18 .
duckham et al . showed that males over 65 had significantly greater rates of outdoor falls
while engaging in recreational or vigorous activities . in turn , older females tended to have
significantly higher rates of indoor falls , which more often proved to be injurious19 .
this study found a correlation between
the number of falls in the 12 months before the study and the score for the tug test
( p<0.001 ) , followed by poma ( p<0.001 ) and olst ( p<0.01 ) .
king et al . investigated
patients with parkinson s disease , who might have difficulty turning over , and also found
tug to be superior to poma and bbs20 .
on
the other hand , chiu et al . compared 4 functional tests , i.e. , bbs , poma , the elderly
mobility scale ( ems ) , and tug , in fallers and non - fallers and concluded that bbs proved to
be the most reliable in discriminating between fallers and non - fallers21 .
some authors are of the opinion that standing balance is
only one of the myriad competencies that contribute to an individual s ambulatory ability .
bbs evaluates the dynamic aspects of balance , and thus it could be used to influence
decisions about an individual s ambulation status or serve as targets for intervention
strategies13 , 14 .
regardless , bbs should be combined with other balance tests in
patients after stroke13 .
sibley et al .
showed that in canada 70% of physiotherapists use olst and bbs to evaluate balance , whereas
tug is used by only 56.9% of physical therapy practitioners , mainly due to lack of time
during their work and lack of sufficient knowledge22 .
the use of balance tests in their work also depends on the goal of
such an assessment .
tug evaluates the basic level of activity , whereas bbs supplies
information about more complex balance - related problems .
barry et al . , in a systematic
review and meta - analysis , showed that tug has limited ability to predict falls among
community - dwelling older adults , and should not be used alone to identify individuals at
high risk of falling23 . on the other
hand , zasadzka et al .
the abovementioned
tests will enable physiotherapists working with elderly residents of nursing homes to
identify individuals who require prophylactic measures to prevent falls , including
introduction of exercises that improve gait , balance , muscle strength , and coordination .
according to schoene et al .
, sensorimotor training with pneumatic compressors , balance
platforms , and diversified exercises , including balance and aerobic exercises , should be
included25 .
based on the findings of
the present study , it seems advisable to use 2 or more tests to assess the risk of falling
in elderly residents of nursing homes . as indicated by the results , tug and poma
are the
most useful tools to screen for balance and gait impairment in this population . | [ purpose ] the risk of falls in the elderly is an important
public health problem .
suitable tests may help detect those at risk of falling .
this study
determined which balance test for older adults generates the most reliable results in
terms of fall risk assessment , based on the number of falls over the last 12 months .
[ subjects and methods ] a total of 153 individuals ( 31 males , 122 females , aged 76.67
8.3 years ; median 76.5 , range 6594 ) were investigated .
the subjects were subdivided
between fallers ( a fall over the last 12 months ) and non - fallers ( no falls over the last
12 months ) .
all participants were assessed with the following : barthel scale , mini - mental
state examination , timed up and go , tinetti performance - oriented mobility assessment ) ,
berg balance test , and one - legged stance test . [ results ] statistically significant
differences were detected between fallers and non - fallers in tug , poma , bbs , and olst
scores .
the number of falls correlated positively with the results for tug , poma , and
olst .
[ conclusion ] tug and poma were the most useful screening tests for balance and gait
impairment in elderly nursing home residents .
two or more tests should be performed for
more precise assessment of the risk of falling . |
to analyse the cytomorphological features of keratinocytes in smears obtained from the oral mucosa of tobacco users and from oral squamous cell carcinoma lesions .
oral smears were obtained from clinically , normal appearing mucosa of oral squamous cell carcinoma patients ( n=20 ) and from the mucosa of smokers ( n=20 ) , and apparently healthy individuals ( n=20 ) were used as controls .
one - way anova ( analysis of variance ) was used for comparing the parameters among multiple groups and tukey - hsd test was used to compare the mean values between groups .
the mean nuclear area of keratinocytes from the mucosa of tobacco users was 46 2.57 and that of the oral squamous cell carcinoma lesion was 81.54 4.31 . while there was a significant ( p=0.001 ) reduction in the cellular area of keratinocytes from oral squamous cell carcinoma lesion when compared with those from oral smears of tobacco users .
cytomorphometric analysis of keratinocytes can serve as a useful adjunct in the early diagnosis of oral squamous cell carcinomas . | aimto analyse the cytomorphological features of keratinocytes in smears obtained from the oral mucosa of tobacco users and from oral squamous cell carcinoma lesions.methodologyoral smears were obtained from clinically , normal appearing mucosa of oral squamous cell carcinoma patients ( n=20 ) and from the mucosa of smokers ( n=20 ) , and apparently healthy individuals ( n=20 ) were used as controls .
the smears were histochemically stained and cytomorphological assessment of the keratinocytes was carried out .
one - way anova ( analysis of variance ) was used for comparing the parameters among multiple groups and tukey - hsd test was used to compare the mean values between groups.resultsthe mean nuclear area of keratinocytes from the mucosa of tobacco users was 46 2.57 and that of the oral squamous cell carcinoma lesion was 81.54 4.31 .
while there was a significant ( p=0.001 ) reduction in the cellular area of keratinocytes from oral squamous cell carcinoma lesion when compared with those from oral smears of tobacco users.conclusioncytomorphometric analysis of keratinocytes can serve as a useful adjunct in the early diagnosis of oral squamous cell carcinomas . |
the differentiation of nave cd4 t cells into effector t helper ( th ) cells is induced by their t cell receptor and costimulatory molecules in the presence of other cytokines .
it is that these cytokines and transcriptional factors ultimately determine the differentiation of cd4 th cells into distinct subsets .
initially , cd4 th cells were identified as having two subsets , th1 and th2 cells .
th1 cells produce high levels of ifn- and express the transcriptional factor t - bet , which protect the host against intracellular pathogens .
th2 cells express gata-3 and produce il-4 , il-5 , il-9 , and il-13 which are mainly involved in protection against parasitic helminthes .
recently , new subsets of effector th cells that express different transcriptional factors and produce distinct cytokines have been discovered , including t regulatory ( treg ) cells , th17 cells , follicular helper t cell ( tfh ) , and th9 cells [ 4 , 5 ] .
treg cells are characterized by the production of il-10 and tgf- as major cytokines and expression of forkhead box p3 ( foxp3 ) as transcriptional factor , which control immune response and maintain immune tolerance .
th17 cells are characterized by the production of il-17a ( also known as il-17 ) , il-17f , and il-22 as signature cytokines and expression of retinoic acid - related orphan receptor gamma t ( rort ) as master transcriptional factor [ 79 ] .
these cytokines play a critical role in host defense against extracellular pathogens such as bacteria and fungi and many autoimmune diseases , including psoriasis , rheumatoid arthritis ( ra ) , inflammatory bowel disease , uveitis , and multiple sclerosis [ 1113 ] .
fully human monoclonal antibodies ( mabs ) against il-17 ( ixekizumab and secukinumab ) and il-17 receptor a ( il-17ra ) ( brodalumab ) have rapidly reduced clinical symptoms in patients with psoriasis [ 1418 ] . however , in a phase ib study on methotrexate - resistant ra patients , brodalumab did not improve disease symptoms . in a phase ii study ,
furthermore , treatment of patients with crohn 's disease with secukinumab not only showed no good responses , but also worsened disease in some patients .
since th17 cells also produce other cytokines such as il-17f and il-22 which are potent inflammatory mediators , targeting th17 cells may provide a better efficacy in these clinical conditions .
th17 differentiation requires the master transcriptional factor , rort , which is induced by activation of nave cd4 t cells in the presence of inflammatory cytokines , such as il-6 , tgf- , il-21 , il-1 , and il-23 .
mice deficient in rort have reduced th17 differentiation and are resistant to experimental autoimmune encephalomyelitis ( eae ) .
the critical role of rort in the generation of th17 cells provides a unique opportunity to develop novel therapeutics targeting th17 cells .
given the fact that cytokines of il-17 family are important in host defense and they are also produced by other immune cells other than th17 cells , it is highly desirable to target the pathogenic th17 cells .
the disadvantage of mabs targeting individual il-17 cytokines is that it does not discriminate the cellular source of il-17 and therefore poses potential adverse effects from blocking il-17 activity produced for host defense .
moreover , the effector cytokines of th17 cells include il-17a , il-17f , and il-22 which are all to be blocked to abrogate th17 cell activity which is a challenging task for individual mabs .
recently , small molecules targeting rort have been identified , which not only suppress th17 differentiation and il-17 production , but also reduce the severity of animal models of autoimmune diseases .
in addition , recent advancement in technology of engineering nucleic acid enables a targeted delivery of small interference rna ( sirna ) or short hairpin rna ( shrna ) using aptamers which serve as vehicle to guide sirna or shrna to target cells .
these two classes of agents , which are nonmonoclonal antibody or fusion protein based , are emerging to be useful in targeting th17 cells rather than merely blocking individual cytokines .
small molecules directly interact with rort to block its activity while sirna / shrna specifically inhibits rort gene expression .
before the identification of th17 cells , t cell mediated autoimmunity was believed to be mediated by th1 cells . indeed , t - bet deficient mice were resistant to eae , and polyclonal antibody targeting il-12 was an effective therapy for eae and cia . however , the later studies provided contradictory results that ifn- and ifn- receptor deficient mice , as well as mice that lack il-12p35 , were not protected from eae but developed rapidly progressing disease [ 24 , 25 ] . furthermore , ifn- knockout mice develop severe eae and convert resistant strain of mice to be highly susceptible to collagen - induced arthritis ( cia ) [ 26 , 27 ] .
the discovery of il-23 , a cytokine which is composed of a unique p19 subunit and a p40 subunit which is shared with il-12 , provided us with novel insights .
it was il-23 , not il-12 , that was critical for the induction of eae and cia [ 29 , 30 ] . moreover , il-23 failed to induce ifn- but instead expanded il-17-producing t cells . when il-17-producing t cells induced by il-23 were adoptively transferred into nave wild - type mice , eae developed .
il-23p19-deficient mice were resistant to eae due to lack of il-17-producing t cells [ 29 , 30 ] .
these studies led to il-17-producing t cells to be described as a distinct th cell subset , which was named th17 cells [ 7 , 8 ] .
differentiation of th17 cells is induced by activation of nave cd4 t cells in the presence of inflammatory cytokines . transforming growth factor- ( tgf- )
is a regulatory cytokine which has multiple effects on t cell development , homeostasis , and tolerance .
tgf- not only induces nave precursors into foxp3-expressing inducible treg ( itreg ) , but also plays a crucial role in the generation of th17 .
however , tgf- alone is not capable of the induction of th17 cells development . unlike th1 , th2 , and itreg cells , which only require a single cytokine for their generation
recent studies found that combination of il-6 and tgf- was the essential cytokine - mix of inducing nave t cells to develop th17 cells [ 3234 ] .
il-6 is able to inhibit tgf--driven induction of foxp3 in nave t cells and instead leads to strong induction of il-17 .
furthermore , il-21 together with tgf- is also able to induce the differentiation of th17 cells . during the initial th17 differentiation ,
il-6 induced il-21 acting as a positive amplification loop to enforce th17 differentiation [ 35 , 36 ] .
il-21 was shown to be able to replace il-6 at least in vitro . in the absence of il-6 , il-21 together with tgf- was able to inhibit the development of itreg and to promote the differentiation of th17 cells . in vivo , however , the role of il-21 in the induction of th17 cells remains controversial .
it had been reported that the absence of il-21 or il-21r had no significant difference on the development of th17 cells [ 38 , 39 ] .
however , il-23 receptors are absent on nave t cells , so il-23 is not involved in the initiation of th17 cells , but expands an existing population of effector th17 cells . without il-23 , activated cd4 t cells in the presence of il-6 plus tgf- were able to produce high amounts of il-17 but did not fully develop into pathogenic th17 cells .
the treatment with neutralizing il-23p19 specific antibody not only inhibited the development of eae but also ameliorated eae after the onset of disease .
ustekinumab , a mab against il-23/il-12p40 , has shown a marked efficacy in clinical studies involving psoriasis patients .
ustekinumab also has shown increased clinical responses in patient with tumor necrosis factor- ( tnf- ) refractory crohn 's disease .
in contrast to mice , combination of il-6 and tgf- is not capable of inducing human th17 differentiation . instead of
tgf- , il-1 together with il-6 or il-23 was reported to upregulate rort and induce il-17 production from cd4 t cells isolated from human peripheral blood , suggesting a fundamental difference in the biology of human and mouse th17 cells .
the differentiation of th17 cells is initiated by the combined signals of activated tcr and cytokine receptors .
these signals then induce specific transcription factors responsible for the expression of th17 cell specific genes such as il17 and il17f .
multiple transcription factors have been shown to be important for the development of th17 cells , including rort , stat3 , irf4 , batf , and runx1 .
ror is the member of retinoic acid nuclear receptor superfamily containing a ligand - binding domain ( lbd ) .
usually , ligand binding to the lbd of ror leads to conformational change and transcriptional activity .
the ror subfamily has three members in mammals : ror , ror , and ror .
the ror has two different isoforms : ror and rort , which are encoded by the rorc gene and have difference only at their n terminus .
rort is a splice variant of ror expressed in t cells . unlike ror , which is expressed in many tissue such as heart , kidney , liver , lung , brain , and muscle ,
rort is an important molecule to regulate gene expression during the development of t cells and the formation of secondary lymphoid organ [ 5153 ] .
rorc gene knockout mice exhibited that cd4cd8 thymocytes showed early apoptosis , and lymph nodes , peyer 's patches , and lymphoid tissue inducer ( lti ) cells failed to develop [ 52 , 53 ] . in vitro , with the absence of rorc in cd4 t cells , il-17 expression was greatly decreased under th17 polarizing conditions .
conversely , overexpression of rort in nave cd4 t cells was sufficient to induce the expression of il-17 , il-17f , and il-22 .
rort is necessary for the expression of il-17 as well as the differentiation of th17 in mouse and human cd4 t cells [ 9 , 54 ] .
the number of th17 cells was markedly reduced and the disease severity of eae alleviated in rorc - deficient mice .
the role of rort is similar to transcription factors such as t - bet and gata3 in th1 and th2 differentiation , respectively , and therefore rort has been considered to be a master transcriptional factor for th17 differentiation .
rort promotes il-17 expression by directly binding the promoter region of il17 gene at multiple sites [ 9 , 55 , 56 ] .
another related retinoic acid nuclear receptor , ror , is also expressed in th17 cells both in vitro and in vivo .
however , mice deficiencies in rora and rorc markedly impaired th17 generation and completely protected mice from eae .
it is demonstrated that ror and rort acts as synergy in regulating th17 cell gene expression . besides ror and rort ,
the transcription factor signal transducer and activator of transcription 3 ( stat3 ) , which is preferentially activated by il-6 , il-21 , and il-23 , is capable of inducing rort and regulating th17 cells development [ 58 , 59 ] .
deficiency of stat3 in cd4 t cells impaired th17 differentiation in vivo , and overexpression of a constitutively active stat3 could increase il-17 production [ 58 , 60 ] .
stat3 might affect the production of il-17 by increasing the expression of rort and ror [ 57 , 58 ] .
furthermore , stat3 also binds directly to the il17 and il21 promoters and leads to the expression of il-17 and il-21 [ 61 , 62 ] .
transcription factor interferon regulatory factor 4 ( irf4 ) also has a certain role in th17 differentiation , which was previously associated with gata-3 expression in th2 differentiation .
irf-4 deficient t cells failed to upregulate rort in response to il-6 plus tgf- and did not differentiate into th17 cells , suggesting that irf4 might also cooperate with rort to induce th17 differentiation .
in addition , batf , a member of the ap-1 transcription factor family , and runx1 , a member of runx1 transcription factor , are also important for th17 differentiation [ 66 , 67 ] .
as mentioned above , rort , stat3 , irf4 , batf , and runx1-deficient mice show an impaired th17 generation and an attenuated susceptibility to the induction of autoimmunity .
targeting these transcription factors might be a possible way to inhibit the development and function of th17 cells .
rort acts as the master transcription factor of th17 differentiation , resulting in an attractive pharmacologic target for the treatment of th17-mediated autoimmune disorders .
by performing a chemical screen with an insect cell - based reporter assay , the cardiac glycoside digoxin was identified as a specific inhibitor of rort transcriptional activity .
in addition , digoxin was effective in attenuating eae in mice and in delaying the onset and reducing disease severity in a rat model of adjuvant - induced arthritis [ 6870 ] .
digoxin was toxic for human cells at high doses , but its synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene were nontoxic and specifically inhibited the induction of il-17 in human cd4 t cells .
these data indicate that derivatives of digoxin might be used as chemical templates for the development of targeting rort therapeutic agents that attenuate inflammatory th17 cells function and autoimmune disease .
using a cell - based gene rort and control reporter assay , a small molecule library comprising 300,000 compounds was screened at the nih chemical genomics center ( ncgc ) , a series of diphenylpropanamide compounds as a selective rort inhibitor , including a highly potent compound ml209 ( also known as compound 4n ) .
huh and colleagues found that compound 4n inhibited transcriptional activity of rort , but not ror , in cells .
like digoxin , compound 4n selectively inhibited murine th17 differentiation without affecting the differentiation of nave cd4 t cells into other lineages , including th1 and regulatory t cells . moreover , compound 4n suppressed rort - induced expression of il-17 in human t cells .
this report demonstrates that compound 4n might serve as a valuable pharmacological agent to inhibit rort transcriptional activity and th17 differentiation . using the liver x receptor ( lxr ) agonist t0901317 scaffold as a lead compound ,
griffin and burris developed a derivative , sr1001 , which was devoid of all lxr activity yet retained its ability to suppress the transcriptional activity of ror and ror .
sr1001 not only is high - affinity synthetic ligand that is specific to both ror and ror , but also inhibits th17 cell differentiation and function .
sr1001 binds specifically to the lbd of ror and ror , inducing a conformational change within the lbd , resulting in suppression of the receptors ' transcriptional activity . by suppressing il-17 gene expression and protein production ,
furthermore , sr1001 inhibited the expression of cytokines in murine or human th17 cells and effectively reduced eae severity in mice .
therefore , sr1001 and its derivatives may represent a novel drug to treat not only th17-mediated autoimmune diseases , but ror - mediated metabolic diseases as well . by modifying the sr1001 scaffold ,
, sr2211 can specifically inhibit the transcriptional activity of ror , but not ror. in cotransfection assays , sr2211 suppresses transcription activity in both gal4-ror lbd and full - length ror contexts .
furthermore , sr2211 could result in suppression of gene expression and production of il-17 in el-4 cells .
these data strongly suggest that sr2211 is also a potent and efficacious ror mediator and represses its activity .
moreover , sr2211 suppressed inflammatory t cell function and th17 cell differentiation and markedly reduced joint inflammation in mice with cia .
it is shown that sr2211 has the potential utility for the treatment of th17-mediated autoimmune disorders .
ursolic acid ( ua ) , a small molecule present in herbal medicine , was identified by screening a small chemical library . in treatment with ua ,
the function of rort was inhibited selectively and effectively , and il-17 expression was greatly decreased in developing and differentiated th17 cells . in addition , ua ameliorated eae in mice .
the results thus indicate that ua might be a valuable drug candidate and can be used for developing treatments of th17-mediated inflammatory diseases . using a fluorescence resonance energy transfer ( fret ) assay and two - cell line reporter assay ( il-17f promoter and ror-lbd promoter assays ) ,
a proprietary small - molecule library was screened and several compounds binding to rort were identified .
tm920 and tm778 were identified as highly potent and selective rort inhibitors [ 77 , 78 ] . in vitro , tm920 and tm778 suppressed th17 development and inhibited il-17 production from differentiated th17 cells .
furthermore , tmp778 has increased potency and specificity for th17 differentiation , resulting in blockade of nearly all th17 signature gene expression .
importantly , tmp778 displays no activity against any of the other 24 nuclear receptors tested , including ror and ror , so tmp778 has very limited effects on the expression of other genes .
tmp778 potently impaired the il-17 production not only by human cd4 th17 cells , but also by human cd8 tc17 cells , memory cd4 t cells , and pbmcs .
tmp778 also blocked il-17 production by skin mononuclear cells of psoriasis patients and significantly impaired expression of th17 signature gene from psoriasis patients . in vivo , tmp778 suppressed imiquimod - induced cutaneous inflammation and eae [ 77 , 78 ] .
although the specific rort inverse agonist , tm778 , may have good rort target effects and low off - target effects , unexpected toxicity may occur in nonimmune cells and tissues ( see below ) ; in particular , it required a relatively higher dose of tn778 to exert its function .
another rort inhibitor , gsk805 , is proved to be more potent than tm778 and can be orally administered .
gsk805 could efficiently ameliorate the severity of eae and strongly inhibited th17 cell differentiation in the central nervous system .
it is interesting but unexpected that tmp778 and gsk805 were able to induce rort biding to gata3 and led to an increase of gata3 mrna and protein expression .
the apparent transactivation of gata3 by rort may partially explain the inhibition of th17 cell signature gene expression by tmp778 or gsk805 .
these compounds target rort , which inhibit the transcriptional activity of rort by binding to rort lbd , a domain present in both ror and rort .
these compounds not only inhibit th17 cell differentiation and il-17 production , but also have shown variable levels of efficacy in eae and cia studies .
therefore , these compounds may serve as novel attractive drugs to treat th17-mediated autoimmune disorders .
however , we should note that ror is broadly expressed in many human tissues such as heart , kidney , liver , lung , brain , and muscle , so ror/rort inverse agonists might induce toxicity via inhabitation of ror in nonimmune tissue .
thus , in order to treat th17-mediated autoimmune disorders , it is necessary to develop a specific strategy to only inhibit ror/rort transcriptional activity in immune cells , especially cd4 t cells .
recently , rna interference ( rnai ) technology provides a promise for studying basic t cell biology and for developing potential t cell targeted therapeutics .
however , efficient delivery of small interference rna ( sirna ) into primary t cells remains a major hurdle of sirna - based therapy .
emergence of cd4 aptamers , which specifically bind cd4 t cells and efficiently deliver various biomolecules into these cells , makes it possible to target rort and il-17 production in cd4 th17 cells with rnai technology . here
we will discuss the advantage of aptamer - sirna and contemplate whether cd4 aptamer - rort shrna chimeras would be beneficial to inhibit th17 differentiation in human t cells .
aptamers , nucleic acid - based ligands , are small single - stranded dna or rna oligonucleotides that are produced in vitro via a process known as systematic evolution of ligands by exponential enrichment ( selex ) [ 81 , 82 ] . in the selex process , aptamers are selected from a large pool ( > 1 10 ) of single - stranded oligonucleotides with random sequences [ 83 , 84 ] . after the incubation of the random aptamers pool with the target , followed by repeated cycles : the fixation of region containing binding , pcr or rt - pcr amplification , and modification of restriction endonuclease , aptamers with high affinity with their corresponding ligands are cloned . with the technological improvement in the selex process
, researchers can isolate aptamers from not only a protein target but also a complex mixture including cell - surface proteins and human plasma in the past decades .
recently , isolation of cell- and receptor - specific aptamers using living cells has been reported [ 8688 ] .
therefore , the power of selex enables one to generate specific aptamers against a molecule , a protein , a cell - surface receptor , and even a cell [ 89 , 90 ] . notably , chemical modifications to aptamers , including sugar modifications ( 2-o - methyl , 2-o - methoxyethyl , 2-fluoro , or lna ) , the phosphate backbone modifications ( phosphorothioate , boranophosphate ) , or the nucleobase moiety modifications ( 4-thiouracil , 2-thiouracil , and diaminopurine ) , have been reported to greatly enhance the nuclease resistance of the aptamer probes [ 91 , 92 ] .
similar to antibodies , aptamers , which are often regarded as nucleic acid antibodies ,
gain entrance to target cells via receptor - mediated endocytosis upon binding to cell surface ligands [ 93 , 94 ] .
however , aptamers are generally nonimmunogenic or low - immunogenic [ 95 , 96 ] , whereas antibodies suffer from immunogenicity , resulting in immune responses in patients .
in addition , the cost of generation of aptamers in vitro is much less than the development process of antibodies [ 93 , 98 ] .
importantly , aptamers can be generated through simple chemical approach in animals or cultured mammalian cells , making them easier to produce for large scale manufacturing that are necessary for clinical use .
the first therapeutic aptamer , antivascular endothelial growth factor ( vegf ) aptamer , macugen ( pegaptanib ) was approved by the us fda for treatment of age related macular degeneration in 2005 .
also , several aptamers are currently undergoing clinical trials [ 92 , 101 , 102 ] .
therefore , aptamers that target various proteins and cells are considered as ideal diagnostic and therapeutic approach for clinical disease , such as cancer , infection , and autoimmune disease [ 99 , 103 ] .
sirna , because of their ability to silence expression of sequence - specific gene [ 106 , 107 ] , has currently been developed as a new strategy in treatment of human disease .
however , it is a big challenge to efficiently and safely deliver sirna into difficult - to - transfect primary t cells by conventional transfection methods . for instance ,
electroporation and nucleofection cause excessive cell death and may require preactivation of t cells and electrical apparatus [ 108 , 109 ] .
chemically modified synthetic sirna with acell agents can be transfected into primary t cells ; however , they are needed to incubate with t cells for longer time and only a small portion of t cells are transfected .
the most disappointing defect of these methods is that it is difficult for them to be used in vivo .
retroviral vectors carrying shrna cassette are able to effectively infect and enter t cells and make the shrna to stably be expressed for the lifetime of the cells in vitro and in vivo [ 111 , 112 ] .
however , applying retroviral vectors in vivo gives rise to the danger about malignant transformation , which limits the viral vector transfection .
nanoparticles are effective to deliver sirna into t cells , but the delivery is not t cell specific .
recently , a method that uses a fusion protein composed of a cell - target antibody fragment joined to a protamine peptide that binds nucleic acids has been reported for cell - specific sirna transfection of immune cells [ 114 , 115 ] .
sirnas mixed with the fusion protein can silence gene expression in cells , both in vitro and in tissues .
however , antibody - based fusion proteins are expensive to manufacture , are potentially immunogenic , and are unsuitable for clinical use .
hence , an effective sirna delivery system in vivo for targeting t cells has to be developed for treatment of t cells - mediated human disease .
because aptamers can enter target cells via endocytosis and maintain stability after endocytosis , aptamers have been developed as guiding moieties for both drug delivery and nucleic acid transport vehicles such as sirna and shrna .
aptamer sirna chimeras , composed of an sirna / shrna fused to an aptamer , provide an attractive alternative for in vitro and in vivo gene knockdown .
the aptamer portion of the chimeras binds to a cell - surface receptor such as prostate surface membrane ag ( psma ) , cd4 , whereas the sirna portion targets the overexpressed signaling molecules or regulatory nucleic acids , resulting in inhibition of cell proliferation and differentiation .
aptamer - sirna chimeras ( asics ) efficiently transfect and knock down gene expression in cells bearing the surface receptor recognized by the aptamer .
the psma aptamer - sirna chimeras targeting psma silenced target gene expression in prostate cancer mouse xenografts .
asics containing an aptamer targeting hiv - gp120 inhibit hiv replication in already infected cells in vitro [ 119 , 120 ] and in vivo .
cd4-asics bearing sirnas that recognized hiv gag and vif or host ccr5 were specifically taken up by cd4 cells , knocked down genes expression , and inhibited hiv infection in primary cd4 t cells and in the female genital tract of humanized mice [ 122 , 123 ] and at the same time do not activate lymphocytes or stimulate innate immunity [ 122 , 123 ] . moreover
, the chimeras do not bind to or function in cells that do not express cd4 , such as cd3 , cd8 , and cd45 .
thus , aptamer - facilitated cell specific delivery of sirna / shrna represents an attractive novel approach for efficient rnai delivery .
cd4-asics overcome the hurdle of in vivo sirna delivery to the immune cells and hold a promise to study immune responses and develop therapeutics in autoimmune diseases .
cd4 aptamers that bind surface cd4 can be applied as t helper cell - specific delivering vehicles .
cd4-asics bearing sirnas or shrna targeting rort might suppress th17 differentiation and treat th17-mediated autoimmune diseases .
we selected cd4 rna aptamers ( 86 nucleotides in length ) that delivered rort - shrna ( figure 1 ) into cd4 t cells and investigated its efficacy in suppressing th17 cell differentiation and il-17 production in human cd4 t cells in vitro .
chemical modifications of nucleotides 2-f - dctp and 2-f - dutp were done to enhance the nuclease resistance of the aptamer chimeras . in vitro using fluorescent microscope and flow cytometric analysis ,
cy3-labeled cd4 aptamer - rort shrna chimeras ( cd4-ashr - rort ) ( 133 nucleotides in length ) were shown to enter into human cd4 t cells but not cy3-labeled mock cd4-ashr- rort . in vitro expression of rort is significantly and specifically diminished by cd4-ashr - rort in a concentration - dependent manner in human cd4 t cells compared with control cd4 aptamers .
consistent with decreased rort , cd4-ashr - rort displayed a concentration - dependent inhibition of il-17a release from cd4 t cells and intracellular il-17a staining in cd4 t cells , while mock cd4-ashr - rort and cd4-ashr - scrambled control have no impacts .
this study indicates that intracellular delivery of cd4-ashr - rort could target rort and manipulate th17 cell differentiation and il-17 production in cd4 t cells .
additionally , cd4-ashr - rort does not significantly impact the expression of th1 and th2 lineage transcription factors t - bet and gata-3 in pmbcs .
consistent with these , synthesis of ifn- and il-4 in pbmcs is not changed by cd4-ashr - rort .
these suggest that cd4-ashr - rort chimeras keep its specificity to target rort gene and th17 cells .
thus , it is of interest to explore the use of cd4-ashr - rort chimeras in animal and clinical trials of autoimmune diseases .
in conclusion , th17 cells and their signature cytokines play crucial roles in the pathology of autoimmune and inflammatory diseases .
targeting il-17 or il-17r has shown clinical efficacy in psoriasis but not many other autoimmune disease such as ra and crohn 's disease .
in contrast to blocking a single effector cytokine , targeting th17 lineage provides promising therapeutic to impact multiple inflammatory cytokines .
first attempts to target th17 lineage are targeting rort , the master transcriptional factor of th17 lineage , via small molecule inverse agonists .
several small molecules are shown to have potent suppressive effects on th17 cells and their cytokines and have therapeutic efficacy in animal models of autoimmune diseases .
aptamer mediated delivery of sirna / shrna specifically against rort offers another strategy to target th17 cells . by replacing the shrna for targeted genes such as gata3 , t - bet , and stat3 ; this cd4 aptamer may be used as a universal tool to introduce sirna or shrna into cd4 t cells to manipulate function of various th cells .
further animal and clinical trials of cd4-ashr - rort chimeras are necessary to evaluate the beneficial outcomes in autoimmune diseases . | t helper 17 ( th17 ) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- ( il- ) 17 , il-17f , and il-22 .
anti - il-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis ( ra ) and crohn 's disease .
alternative agents targeting th17 cells may be a better way to inhibit the development and function of th17 cells than antibodies of blocking a single effector cytokine .
retinoic acid - related orphan receptor gamma t ( rort ) which acts as the master transcription factor of th17 differentiation has been an attractive pharmacologic target for the treatment of th17-mediated autoimmune disease .
recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting rort .
chemical screen technology identified several small molecule specific inhibitors of rort from a small molecule library .
systematic evolution of ligands by exponential enrichment ( selex ) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library . in this review ,
we highlight the development and therapeutic potential of small molecules inhibiting th17 cells by targeting rort and aptamer mediated cd4 + t cell specific delivery of small interference rna against rort gene expression to inhibit pathogenic effector functions of th17 lineage . |
probiotics have gained significant importance in the last few decades for their health promoting roles in the prevention and prophylaxis of various gut associated disorders , urogenital , and respiratory infections.1 they have also been shown to positively affect the host immune system through immunoglobulin production , and trigger cell - mediated immune responses as a frontline of defense.2,3 probiotics are described as live microorganisms which when administered in adequate amounts confer a health benefit on the host.4 the therapeutic potential and antimicrobial spectra of probiotics is a complex and a multifactorial process which involves the production of organic acids , hydrogen peroxide,5,6 bacteriocins , bacteriocin - like inhibitory substances,7,8 short - chain fatty acids ( scfas ) , conjugated linoleic acid ( cla ) , and -amino butyric acid ( gaba)912 ( figure 1 ) .
many studies have elucidated the health benefits and clinical effects of probiotics in gastrointestinal abnormalities including irritable bowel syndrome , irritable bowel disease , gastric ulcers , rotavirus , traveler s and antibiotic - associated diarrhea , colorectal cancer , and in the alleviation of lactose malabsorption.13,14 recently , probiotics have undergone scientific scrutiny for their potential in reducing the risk of cardiovascular diseases ( cvds ) and they have been shown to be effective in improving the health conditions among the tested subjects with cardio - associated diseases.1519 the estimated total number of adults with hypertension worldwide during the year 2000 was 1 billion and predicted to rise to 1.58 billion by the year 2025.20 certain probiotic strains such as lactobacilli and bifidobacteria can effectively produce scfas , cla , gaba,12,2123 and ace inhibitory peptides , which have shown potential hypotensive effects.2426 growing public awareness of diet - associated health issues has fueled the functional foods concept , foods that provide specific health benefits over and above their nutritional value,10 which can be produced under controlled fermentation conditions or enriched with added nutrients .
functional foods enhance the overall nutritional status with added vitamins and minerals including probiotics and their biogenic metabolites.27 many studies have shown the health benefits associated with the consumption of functional foods incorporated with probiotics , such as cheese,28 milk , fermented milk products,21,29,30 and non - dairy beverages.31 in this review we emphasize the biochemistry of hypertension , diet associated disorders , and the role of probiotics in controlling elevated blood pressure ( bp ) levels , nutritional programming , and the application of probiotic cultures in reducing the onset and development of cvd .
metagenomics has revolutionized the field of microbiology by paving the way for culture - independent assessment and exploration of microbial communities residing in complex ecosystems .
the mammalian gastrointestinal tract ( git ) is among the most densely populated with complex microbial communities and the colon merely harbors a load of ~10 cells / host.32 the human intestinal microbiota plays a pivotal role in the host s digestive process , gut maturation , epithelial cell development , and a regulatory switch to innate immunity , thereby contributing to host health33 and many more functions yet to be revealed which are associated with the gut microbiota .
the metabolic capacity of the core gut microbiome is so prodigious that it has been considered as the virtual organ of the git,34,35 and it is known to influence human health and disease susceptibility in different ways .
microbial colonization of the host starts during birth , and the composition of the microbiota widely varies throughout host development.36,37 a variety of factors during and after birth , such as mode of birth ( vaginal versus cesarean - section ) , feeding ( breastfed or bottle fed ) , and antibiotics play a significant role in shaping the gut microbiota,38 which reflects their proximity in the health - disease state equilibrium during the growth and development of their host.39 furthermore , the possible role of early colonizers during infancy and changes in their relative composition of the gut microbiota during childhood that can lead to the accumulation of body weight and obesity has been elucidated.40 a recent study revealed the presence of unique microbiome in the human placenta and its resemblance to their oral microbiome,41 suggesting that it is imperative to map the microbial networks residing at different niches to understand their role in health and disease .
recent studies have revealed that many health maladies are as a result of significant perturbation in core gut microbial communities , and many parameters such as host - microbe crosstalk which are intrinsically linked to the microbial ecology and gut functionality .
the influence of git microbiota composition and their possible link to obesity,42 diabetes,43,44 neural disorders,45,46 brain development,47 insulin resistance , and other metabolic disorders48 have been well documented .
furthermore , an aberration in the core - gut microbiota in tlr-5 deficient mice leads to the development of metabolic syndrome.49 recently , more evidence has been accumulated by deciphering the role of gut microbiota in developing cvd .
for example , gut microbiota metabolizes specific dietary nutrients which belong to the trimethylamine group ( eg , choline , phosphatidylcholine , and l - carnitine ) resulting in the formation of the pro - atherogenic compound called trimethylamine - n - oxide ( tmao),50,51 and the carnitine metabolic- pathway associated gene clusters are identified in the genomes of human microbiota.52 altogether , compositional sequencing approaches coupled with transcriptomics studies extrapolated the microbe - host interaction and their interplay among various metabolic and biochemical pathways at the molecular level . however , information on the key role - players is still unclear and yet to be determined for modulation or to reprogram the microbial territories to overcome the health ailments .
there are many factors in developing hypertension , such as sedentary lifestyle , lipid and cholesterol metabolism ( obesity ) , sodium sensitivity , personal habits ( alcohol consumption , smoking ) , anxiety , stress , and vitamin d deficiency.22 there is direct evidence of the factors that control bp such as : a well programmed nutritional strategy and lifestyle , maintaining recommended body mass index ( weight loss ) , reduced salt intake , dietary approaches to stop hypertension-type dietary pattern ( vegetarian diet , more fruits , vegetables , and low - fat dairy products ) , increased potassium intake , and moderation of alcohol intake.53,54 it has been demonstrated that the risk of developing cvd was significantly lower in vegetarians when compared to omnivores.5557 in this context , vegetarians had a significantly higher abundance of bacteroides species and lower abundance of prevotella species in their core gut microbiomes when compared to omnivores , and a reduced risk of developing cvds.58 similarly , a study in men ( aged between 4157 years ) , whose diet intake chiefly includes vegetables and fruits showed a reduced risk of developing high bp.59 these studies indicate the importance of dietary habits and their influence on overall health status and git microbiota .
one way of modulating the gut microbiota is by the consumption of probiotics , in particular , products containing lactic acid bacteria . in this context ,
have been extensively studied as probiotic microorganisms , while other groups such as enterococcus , oenococcus , propionibacterium , bacillus , escherichia coli , clostridium butyricum , and some yeast strains , such as saccharomyces boulardii , have also been used . in recent years
, functional foods containing probiotics have become popular within the food industry due to the heightened awareness of consumers toward these health - promoting foods.60 nutritional programming to manipulate the composition of the intestinal microbiota through the administration of probiotics , prebiotics , and or synbiotics ( a combination of probiotic and prebiotic ) continues to receive much attention for preventing or attenuating the symptoms of metabolic - related diseases .
the maintenance of bp homeostasis is a complex process which is carefully regulated by a variety of inputs .
hypertension or bp , defined as systolic blood pressure ( sbp ) above 140 mmhg and diastolic blood pressure ( dbp ) above 90 mmhg , is one of the key risk factors for an individual prone to many diseases including coronary heart disease , cerebral hemorrhage , renal and cardiac failure.22,61,62 bp is controlled by a number of complex biochemical pathways .
typically , the renin - angiotensin system ( ras ) is known to play a key role in bp regulation and sodium metabolism .
in addition to ras , the kinin - nitric oxide system , the neutral endopepti - dase system , and the endothelin - converting enzyme systems have been shown to produce additional vaso - regulatory peptides.63 however , ras has been identified as one of the major controllers of bp among the others identified ; which play a central role in controlling the level of other key vaso - active peptides.63 ace is a carboxypeptidase responsible for the generation of the potent vasoconstrictor angiotensin ii by releasing the c - terminal dipeptide his - leu from angiotensin i , and is also responsible for the inactivation of the vasodilator bradykinin , which gives rise to a net hypertensive effect.63 together these systems produce a wide array of peptides that collectively regulate bp , electrolyte balance , and fluid equilibrium via membrane bound receptors located in different tissues.64 ras comprises of : i ) agt a globular protein which serves as a substrate for ii ) renin an enzyme that catalyzes the proteolytic conversion of agt to angiotensin i ; iii ) ace ( ec 3.4.15.1 ) , a key enzyme of the ras which controls the arterial bp and water - salt equilibrium in the body;65 and iv ) angiotensin ii receptor.66 the inhibition of ace could lead to antihypertension .
recently , the influence of two sensory receptors for scfas ( olfr78 and gpr41 ) in bp regulation has been identified.67 bp is a multifactorial trait which is regulated by multiple biochemical pathways and all the networks are firmly interlinked .
genomes of lactic acid bacteria ( lab ) encode an array of proteolytic cassettes and peptide transporters.68 in general , lab are cell factories for many proteolytic enzymes ( present on cell envelope and intracellular peptidases ) which are involved in the hydrolysis of peptide bonds generating short oligopeptides.69,70 probiotics have been reported to exert ace - inhibitory activity by producing antihypertensive bioactive peptides which are released during protein hydrolysis.10,71 similar to ace - inhibitory peptides , other peptides , casokinins and lactokinins , are also being released during enzymatic proteolysis of milk proteins and microbial fermentations.72 hence , fermented milk products that are rich in bioactive peptides are considered as natural dietary sources to control hypertension .
in addition to that , probiotic cultures with certain traits such as exopolysaccharides,73 cla,74 and gaba production22,75 positively influence the host lipid metabolism and gut microbial compositions ( figure 1 ) .
the scfas produced by gut microbes , in particular propionate modulates bp levels via gpr41 and olfr78 receptors . furthermore
, olfr78 knockout mice with reduced gut microbial biomass upon antibiotic treatment showed elevated bp levels.76 similarly , reduced microbial richness and diversity has been observed in spontaneously hypertensive rats , with an increase in firmicutes / bacteroidetes ratio and decrease in acetate , butyrate - producing microbes,77 clearly indicating that our gut microbiota are master regulators of hypertension .
vitamin d has been identified as one of the key role - players , among others ( vitamin c and e ) .
an insufficient vitamin d level has been observed in 50% of the world s population and hypovitaminosis d leads to the development of hypertension .
furthermore , the antihypertensive effects of vitamin d are mediated by renoprotection , prevention of secondary hyperparathyroidism , vasodilation , suppression of the renin - angiotensin - aldosterone system , and anti - inflammatory effects have also been validated.78,79 studies have shown the association between vitamin d deficiency and elevated bp levels among the individuals tested.78 oral administration of probiotic lactobacillus reuteri national collections of industrial , marine and food bacteria ( ncimb ) 30242 ( cardioviva ) increased serum vitamin d levels by 14.9 nmol / l and the levels of other vitamins ( a , e and -carotene ) were unaffected.80 intensive research is required to understand the vitamin biosynthesis pathways of probiotic bacteria in vivo . such studies aid us in developing live vitamin delivering cultures to combat vitamin deficiencies in the gut microenviroments .
furthermore , probiotic cultures are known to produce b vitamins such as folate ( vitamin b9)81 and vitamin b12,82,83 which could be interesting in cases of vitamin deficiency . until now
only one study had shown the improvement of vitamin d levels upon probiotic administration in human subjects , therefore , there is a need for more clinical trials to support this hypothesis .
the beneficial role of probiotics in improving cardiovascular health and in the reduction of bp can not be ruled out ; in order to confirm this role , more extensive studies are needed to understand the mechanisms underlying probiotic action . in a review by ness et al , some studies have shown an inverse association between plasma vitamin c levels and bp , and a few reported an inverse association with vitamin c intake.84 in a recent meta - analysis , it has been found that a daily dose of vitamin c ( 500 mg ) for a period of 8 weeks significantly reduced dbp by 1.670.72 mmhg.85 in contrast , administration of 500 mg of vitamin c for 5 years had no effect on bp.86 in conclusion , the association between vitamin c and controlling bp remains unclear due to the inconsistent results observed among research studies .
many minerals are involved in controlling bp levels ; among them the major minerals positively involved in bp regulation are potassium , magnesium , and calcium.87 a study has shown that consumption of yogurt containing probiotic strains ( lactobacillus casei , l. reuteri , and lactobacillus gasseri ) containing yogurt increased apparent calcium absorption in growing rats.88 the ability of probiotics and prebiotics to increase micronutrient absorption has been examined in different studies . although the obtained results were not uniform , an increased rate of mineral absorption was noticed in probiotic groups.89 in summary , due to the lack of a large number of studies on probiotics and their associative link with increased vitamin levels and mineral absorption , this area is still unclear and a solid conclusion can not be drawn regarding the role of probiotics with respect to vitamins and minerals .
therefore , it is advisable to monitor the key vitamin and mineral levels in probiotic clinical trials to extrapolate the link between them .
a substantial body of evidence firmly supports the health benefits and clinical effects associated with probiotics and probiotic fermented foods based on in vitro and in vivo studies . in recent years probiotics and their potential role in maintaining cardiovascular and renal health
numerous studies have shown either moderate or significant reduction in the ratios of sbp / dbp ( table 1 ) .
for example , administration of sour milk fermented with lactobacillus helveticus lbk-16h containing bioactive tripeptides ( commercialized as evolus ; valio dairy , helsinki , finland ) for 21 weeks reduced the mean sbp to 6.7 ( 3.0 ) mmhg in 36 hypertensive subjects when compared to the control groups.26 similarly , a mean reduction of sbp 5.2 ( 8.1 ) mmhg and dbp 1.7 mmhg has been recorded in borderline hypertensive men ( aged 2359 years ) given sour milk fermented with l. helveticus and saccharomyces cerevisiae containing tripeptides ( commercialized as ameal s ; calpis food industry , tokyo , japan ) . it has been shown that administration of l. casei ( lex ) cell lysate reduced bp , triglycerides , plasma cholesterol , and glucose levels when compared with the control group.91 in a study , oral administration of probiotic cultures lactobacillus rhamnosus gg and streptococcus thermophilus containing milk along with vegan food significantly improved lipid profiles and controlled the coliforms in the colon of rats.92
l. helveticus ( lbk-16h strain ) fermented sour milk containing ace - inhibitory tripeptides attenuated the development of hypertension in spontaneously hypertensive rats.93 in a study , milk fermented with l. casei strain shirota and lactococcus lactis yit 2027 and enriched with gaba ( 1 mg / ml ) significantly reduced the mean sbp ( 17.44.3 mmhg ) and dbp ( 7.55.7 mmhg ) in mildly hypertensive patients.22 furthermore , a meta - analysis based on 14 randomized placebo - controlled clinical trials has shown that probiotic fermented milk significantly reduced both sbp and dbp in pre - hypertensive and hypertensive subjects.94 tanida et al showed that intraduodenal injection of lactobacillus johnsonii la1 ( 1109 cfu / day ) , or its metabolites , reduced hypertension and renal sympathetic nerve activity in urethane - anesthetized rats .
this study suggests that la1 or its metabolites might lower bp by changing autonomic neurotransmission via the central histaminergic nerves and the suprachiasmatic nucleus in rats.95 in a double - blind , randomized placebo - controlled trial , consumption of a lactobacillus plantarum 299v ( 210/cfu / ml / day ) fermented food product by 36 smokers for 6 weeks significantly reduced sbp ( 134 mmhg , p<0.001 ) .
moreover , significant reductions were also observed in fibrinogen and low - density lipoprotein cholesterol , leptin , il-6 , and f2-isoprostane concentrations , which serve as biochemical markers for lipid peroxidation and oxidative stress.96 lactic acid bacteria are able to metabolize the complex milk protein and aid in the release of short bioactive peptides which have ace - inhibitory activity , thereby contributing to the modulation of hypertension.71,97,98 in another study , fermented soy milk probiotic cocktail ( l. casei , lactobacillus acidophilus , lactobacillus bulgaricus , s. thermophilus , and bifidobacterium longum ) enriched with whey - separated bioactive peptides with high ace - inhibitory activity positively reduced sbp in rats after 8 weeks of oral application.99 earlier studies have shown the link between gut microbiota and tmao levels in developing cvd . in a recent study ,
subjects who received probiotic l. casei shirota ( dose of 6.510 cfu thrice a day ) for 12 weeks showed reduced levels of tmao when compared to the control group.100 even though the level of reduction is not significant , it is noteworthy to explore the beneficial role of probiotics in multiple aspects of improving the overall health status .
altogether these studies support the antihypertensive activity of probiotics and consumption of probiotic fermented foods for improving overall health status and reducing the risk of developing cvds .
it is noteworthy that regulation of hypertension via administration of probiotics is cross - linked with several different mechanisms , such as improving lipid levels , triglyceride levels , bile acid deconjugation , and controlling body mass index ( figure 2 ) .
in addition , an increase in absorption of nutrients , phytoestrogens ( act as vasodilatory factors ) , and reduction in plasma glucose levels may also influence the probiotic effect in bp regulation.17,65 furthermore , this area of research needs to be examined thoroughly in more clinical studies to postulate the effect of probiotics in the regulation of hypertension .
an increasing number of clinical trials supporting the probiotic - dependent attenuation of hypertension and hypercholesterolemia could provide immense support for the application of such cultures to improve cardiovascular health . hence , dietary intervention to correct gut microbiota could be an innovative nutritional therapeutic strategy for hypertension .
the knowledge obtained on probiotic potential against cvds is still at infancy stage and current findings suggest that hypotensive effects of probiotics are very promising and worth exploring to promote cardiovascular health . however , more studies are required for a better understanding of gut microbiota - host crosstalk and biochemical networks underlying control of hypertension .
as bp is interlinked with other metabolic disorders , it is necessary to examine the outcomes in a meticulous manner to get a clear picture of probiotic action against cvds . | gut microbiota play a significant role in host metabolic processes , and recent metagenomic surveys have revealed that they are involved in host immune modulation and influence host development and physiology ( organ development ) .
initially , probiotics are identified as potential therapeutics to treat gastrointestinal disorders and to revitalize the disturbed gut ecosystem .
currently , studies are exploring the potential for expanded uses of probiotics for improving the health conditions in metabolic disorders that increase the risk of developing cardiovascular diseases such as hypertension .
further investigations are required to evaluate targeted and effective use of the wide variety of probiotic strains in various metabolic disorders to improve the overall health status of the host .
this review addresses the causes of hypertension and the hypotensive effect of probiotics , with a focus on their mechanistic action . |
split cord malformations ( scms ) are rare congenital anomalies in which the cord is split over a portion of its length to form a double neural tube in a single dural sac2,5,16,20 ) .
scms associated with a split of the spinal column , spinal bony spurs , myeloceles , myelomeningoceles , lipomas , and dermal sinuses have been reported in the literature10,12,13,22 ) .
we report a rare case of cervicothoracic scm with spina bifida and tethered cord syndrome due to the thickened filum terminale in adult and discuss the anatomical and embryological relationship of the level of the fibrous septum and duplicated level of the spinal cord .
a 34-year - old woman was admitted with neck pain , back pain , and recurrent bilateral leg weakness of 2 months duration . on physical examination
, there was a paresis with 4/5 muscle strength and deep tendon hyperreflexia in both legs .
the radiologic examination revealed mild scoliosis of the thoracic spine , hemi - vertebrae at t6 level , and butterfly vertebral bodies at t11 level .
the admission cervicothoracic mri showed that the spinal cord was divided into two segments from the level of c-7 to t-11 , and there was development the subcutaneous fat within the bifid spine .
the conus was shown to be low - lying , ending at the l5 level ( fig .
a fibrous septum divided the spinal cord and extended to the epidural space and attached to the ventral cortex of the lamina at t9 level ( fig .
2 ) . the patient underwent surgery to prevent progression of the neurologic damage resulting from tethering of the spinal cord .
the t-9 and t-10 spines were bifid and abnormal fat tissue was infiltrated between the bisected spinous process .
surgery revealed a symmetric duplication of the spinal cord above the level of t-10 , which was encompassed by a single dural sac , and the fibrous septum was recognized between the divided hemi - cord at the t-9 level , 10 mm above the cord reunion site ( t-10 ) .
, a l5 and s1 laminectomy was performed to release the tethered cord syndrome by the thickened filum terminale21 ) .
the nerve roots appeared slightly adherent to the dura at the l-5 level , and resection of the thickened filum was made because the cord was tethered at the level of the lesion ( fig .
the patient had an uneventful postsurgical recovery . at the 7-year follow - up examination ,
the patient had a normal neurologic examination , with no complaints of episodic paraparesis or back pain .
a 34-year - old woman was admitted with neck pain , back pain , and recurrent bilateral leg weakness of 2 months duration . on physical examination
, there was a paresis with 4/5 muscle strength and deep tendon hyperreflexia in both legs .
the radiologic examination revealed mild scoliosis of the thoracic spine , hemi - vertebrae at t6 level , and butterfly vertebral bodies at t11 level .
the admission cervicothoracic mri showed that the spinal cord was divided into two segments from the level of c-7 to t-11 , and there was development the subcutaneous fat within the bifid spine .
the conus was shown to be low - lying , ending at the l5 level ( fig .
a fibrous septum divided the spinal cord and extended to the epidural space and attached to the ventral cortex of the lamina at t9 level ( fig .
2 ) . the patient underwent surgery to prevent progression of the neurologic damage resulting from tethering of the spinal cord .
a laminectomy was performed from the level of t-8 to t-11 . the t-9 and t-10 spines were bifid and abnormal fat tissue was infiltrated between the bisected spinous process .
surgery revealed a symmetric duplication of the spinal cord above the level of t-10 , which was encompassed by a single dural sac , and the fibrous septum was recognized between the divided hemi - cord at the t-9 level , 10 mm above the cord reunion site ( t-10 ) .
, a l5 and s1 laminectomy was performed to release the tethered cord syndrome by the thickened filum terminale21 ) .
the nerve roots appeared slightly adherent to the dura at the l-5 level , and resection of the thickened filum was made because the cord was tethered at the level of the lesion ( fig .
at the 7-year follow - up examination , the patient had a normal neurologic examination , with no complaints of episodic paraparesis or back pain .
scm is a rare spinal anomaly that refers to a longitudinal division of the spinal cord into two divided hemi - cords6,11 ) .
this malformation has been reported to be associated with a split of the spinal column , spinal bony spurs , myeloceles , myelomeningoceles , lipomas , and dermal sinus15,16 ) .
reported the incidence rates for cervical and cervicothoracic locations as 3% and 1% , respectively21 ) .
symptoms related to congenital anomalies occur most commonly in childhood , so scms were initially regarded as a pediatric problem ; but in many patients the diagnosis is not established until symptoms manifest in adulthood1,9,18,19 ) .
embryologically , the spinal cord is formed by the integration of two paramedian notochords along the midline17 ) .
scms are classified as one of two types , according to the unified theory : in type i scm , the hemi - cords are always invested with individual dural sacs and the medial walls of the sacs always ensheath a rigid ( bony or cartilaginous ) midline spur ; and in type ii scm the hemi - cords are always within a single dural sac and the midline septum is always composed of non - rigid fibrous or fibrovascular tissues16,17 ) . based on this classification ,
our case can be classified as type ii because the spinal cord was divided by a fibrous septum at the level of t-9 .
an interesting finding of this case was the relationship between the anatomic location of the fibrous septum and the level of duplication of the scm .
embryologically , scm is caused by a primary neurulation defect . according to the unified theory of embryogenesis proposed by pang et al.17 ) , an abnormal communication ( endomesenchymal tract ) between the ectoderm and endoderm causes regional splitting of the notochord , and each separated notochord induces the surrounding paraxial mesoderm and subsequent dysgenesis of the spinal cord .
thus , the level of the fibrous septum and duplicated level of the spinal cord should be similar .
however , the fibrous septum was located farther above the level of the spinal cord duplication in this case
a low - lying conus and thick filum terminale are related to abnormal retrogressive differentiation of the secondary neural tube with failure of terminal cord involution7,17 ) .
the thickened filum terminale fixed conus and relative ascent of the spinal cord was not complete
. this embryologic background could be the basis for the fibrous septum located proximal to the level of the spinal cord duplication in this case . and
also , anatomical location of the fibrous septum and level of cord duplication of this case could delineate the sequence of spinal cord development .
this anatomic relationship might be helpful to identify the fibrous septum to detether type ii scm patients associated with thick filum terminale .
associated skeletal dysgenesis such as spina bifida , hemi - vertebrae , or a laminar defect might also be helpful to decide the level of detethering surgery .
an awareness of the presence of associated congenital anomalies is surgically important to improve postoperative outcomes and to determine surgical priorities3,8,22 ) for example , if the scm is accompanied by a thickened filum terminale , the septum should first be excised to release the cord .
otherwise , the abruptly released cord will suddenly be pulled upwards , resulting in spinal cord injury below the level of the scm14 ) .
unlike pediatric patients , adult - onset scm may present without neurocutaneous or overt skeletal abnormalities .
a thorough evaluation of associated anomalies is mandatory to make a therapeutic plan for patients with scm .
although it is rare , careful preoperative examination and refined microsurgery may provide good outcome for the patient . | split cord malformations ( scms ) usually present in childhood , and are rarely reported in adults . and also , a cervicothoracic scm associated with tethered cord syndrome has very rarely been reported in the literature .
we report a case of scm associated with tethered cord and spina bifida in an adult .
this report describes the case of a 34-year - old woman who presented for evaluation of neck pain , back pain , and intermittent paraparesis of several months duration . the mri and ct showed a scm at the cervicothoracic level and a fibrous septum at the thoracic level .
she underwent surgery for the scm and tethered cord syndrome , and was followed for 7 years .
patient presented complete recovery in the follow - up .
the authors discuss this unusual lesion and describe the anatomical relationship of the level of cord duplication and fibrous septum . |
vagal nerve blockade ( vbloc ) has been recently studied as a minimally invasive laparoscopic surgery for weight loss and improvement in weight - related conditions such as type 2 diabetes mellitus ( dm2 ) [ 13 ] .
the most frequently performed bariatric surgical procedures for weight loss , roux - en - y gastric bypass and sleeve gastrectomy , produce considerable weight loss but have potentially serious complications and require alterations of the gastrointestinal ( gi ) anatomy that are not acceptable to many patients .
the vbloc device was developed for patients with moderate to severe obesity , as an alternative to conventional weight loss surgery and does not require permanent anatomical alteration .
two recent studies have examined the effect of vbloc on weight loss with a rechargeable device .
the single - arm , prospective vbloc dm2 study showed that at one year subjects with dm2 and body mass index ( bmi ) between 30 and 40 kg / m achieved 25% excess weight loss ( % ewl ) . in the double - blind , randomized recharge trial , using the last - observation - carried - forward ( locf ) analysis method , the vbloc arm achieved 24% ewl ( 9.2% total weight loss [ % twl ] ) at 12 months , which was significantly greater than the 16% ewl ( 6.0% twl ) achieved by subjects in the sham arm implanted with a sham neuroregulator .
both studies showed that vbloc had a low rate of serious device - related complications in the first year .
this report summarizes additional safety and efficacy data from the recharge trial beyond 12 months as patients were unblinded through 18 months of follow - up .
briefly , participants were enrolled at 8 sites in the united states and 2 sites in australia .
participants were eligible for inclusion if their bmi was between 35 and 40 kg / m with one or more obesity - related comorbidities ( i.e. , type 2 diabetes mellitus , hypertension , dyslipidemia , sleep apnea syndrome , or obesity - induced cardiomyopathy ) or had a bmi between 40 and 45 kg / m regardless of comorbidities .
participants with dm2 were limited to 10% of total enrollment so that the weight loss - limiting impact of diabetes would not have an undue impact on study results .
the recharge trial is a 5-year , double - blind , sham - controlled trial comparing vbloc to an implanted sham device .
participants were randomized at implant in a 2 : 1 ratio to vbloc and sham control arms in permuted block sizes of 3 or 6 stratified by clinical site and type 2 diabetes mellitus status .
the surgeon and the surgery support team could not be blinded , so their interaction with participants after randomization was limited until the 12-month blinded period of the trial had elapsed for all participants .
implant of the vbloc device and electrodes requires standard laparoscopic surgery performed under general anesthesia .
electrodes are placed around the anterior and posterior vagal nerve trunks near the gastroesophageal junction , secured with sutures and connected to a rechargeable neuroregulator placed in a subcutaneous pocket on the thoracic side wall .
the neuroregulator is recharged transcutaneously with a transmit coil placed over the neuroregulator connected to a mobile charger .
participants randomized to the sham arm were implanted with a similar neuroregulator that dissipated charge at a rate similar to the active device into a resistor within the sham neuroregulator .
electrodes were not implanted and the vagal nerve trunks were not manipulated . to support the blind , sham patients had the same number of skin incisions to simulate a laparoscopic procedure , but without entering the abdominal cavity .
active and sham neuroregulators were sized identically at 8.6 cm in diameter , 7.1 cm in width , and 1.6 cm thick .
the neuroregulators in both groups were programmed to deliver therapy for at least 12 hours per day ( though no therapy was delivered in the sham group ) .
therapy energy levels were increased over the first month to the desired amplitude of 6 ma , although the amplitude could be adjusted by the follow - up team if the participant felt uncomfortable therapy - related sensations at any time during the trial .
all participants were asked to check their battery level daily and to recharge their battery approximately twice weekly . follow - up visits occurred weekly in the first month , biweekly through month 3 , and monthly thereafter through the second year .
the weight management program typically consisted of a 15-minute educational interaction discussing healthy food choices , physical fitness , and social support .
no specific diet ( e.g. , portion - controlled meals ) or exercise program was prescribed .
related serious adverse events , as defined in the previous publication , through 18 months were classified according to the clavien - dindo classification of surgical complications .
analyses of weight loss data were conducted under the intention - to - treat ( itt ) principle using mixed - effects regression models .
data were analyzed using a linear mixed model with unstructured covariance matrix , treating time ( study visits ) as a categorical variable with time - specific contrasts . under this model , data are treated assuming missingness at random .
since participants were unblinded on a rolling basis after 12 months , two sensitivity analyses were performed to ensure that weight - related trends were not attributable to effects of unblinding .
firstly , a mixed - effects model was fit with a time - varying covariate for unblinding and its interaction with the treatment group to test whether the treatment effect was affected by unblinding of participants .
secondly , the mixed - model was fit to the subset of data for which participants were still blinded .
in this report , results are not reported using the last observation carried forward ( locf ) method , the primary imputation method for the 12-month results , due to the poor statistical properties of locf imputation .
the baseline characteristics of the itt population are summarized in table 1 . a consort diagram through 18 months
is shown in figure 1 . by the 18-month visit , 20 participants ( 12.3% ) in the vbloc group and 13 participants ( 16.9% ) in the sham group
five withdrawals in the vbloc group and 1 in the sham group occurred at implant and have been previously discussed .
after implant in the vbloc group , 2 participants were lost to follow - up , 3 participants withdrew for an adverse event ( pain at the neuroregulator site , heartburn , and pain with therapy , resp . ) , and 10 withdrew for subject decision . in the sham group ,
6 withdrawals were for adverse events ( 2 for pain at the neuroregulator site , rotator cuff pain , irritable bowel syndrome , anxiety , and breast cancer , resp . ) and 6 for subject decision .
the 18-month visit completion rates were 72% in the vbloc group and 55% in the sham group ; however , approximately 80% of participants in the itt population in both groups had weight measurements within 2 months of the 18-month visit , which were incorporated in the statistical analysis of 18-month results .
eleven participants in the vbloc group and 19 in the sham group who did not attend the 18-month visit had attended either their 16- or 17-month visit .
in addition to the revision procedures reported in the first 12 months , there were an additional 3 revisions in the vbloc group between 12 and 18 months .
the revision procedures were uncomplicated and the patients were released within a day of the procedure .
all participants and blinded study personnel remained blinded to randomization assignments until all participants had completed their 12-month visit and the 12-month study database was locked and verified . since the study was enrolled over approximately 7 months , the majority of participants were not unblinded until their 16-month visit .
at 15 months , 85% of subjects in the vbloc group and 90% of subjects in the sham group remained blinded . at the 18-month visit , 27% of vbloc participants and 25% of sham participants were still blinded .
weight loss as both % ewl and % twl over time is shown in table 2 and figure 2 .
for the itt population at 12 months , the estimated mean % ewl was 26% in the vbloc group and 17% in the sham group ( p < 0.001 ) . at 15 months , where 86% of subjects remained under the study blind ,
the estimated mean % ewl was 24% for the vbloc group versus 13% for the sham group ( p < 0.001 ) . at 18 months ,
the estimated mean % ewl was 24% for vbloc and 10% ewl for sham ( p < 0.001 ) .
the corresponding treatment difference between groups increased from 9 percentage points at 12 months ( 95% ci , 414 ) to 13 percentage points ( 95% ci , 818 ) at 18 months .
sensitivity analyses showed that unblinding did not significantly influence the larger treatment effect with vbloc over time as the sham group regained weight ( p = 0.34 for the unblinding by treatment interaction ) .
similarly , when the analysis was restricted to patients who remained blinded throughout 18 months , the estimated mean % ewls were similar to that of the overall sample . at 15 months , the estimated mean % ewl was 26% in vbloc and 13% in sham ( p < 0.001 ) ; at 18 months , the estimated mean % ewl was 21% in vbloc and 8% in sham ( p < 0.001 ) . among the patients completing the 18-month visit ( without imputation ) , the mean % ewl was 25% in the vbloc group and 12% in the sham group ( treatment difference , 13 percentage points ; 95% ci , 621 ) . at 18 months , 54% of vbloc patients achieved at least 20% ewl
compared to 26% in the sham group ( p = 0.002 ) ; 41% vbloc patients achieved at least 25% ewl compared to 17% in the sham group ( p = 0.004 ) .
the adverse event ( ae ) profiles of both treatment groups were similar to that reported at 12 months .
the most commonly reported related aes were heartburn and dyspepsia , abdominal pain , another pain , eructation / belching , and dysphagia .
ninety - eight percent of all aes in the trial were reported as mild or moderate in severity ( versus only 2% reported as severe ) and 79% of events had resolved at 18 months .
most of the related events were transient side effects of therapy and resolved either spontaneously with no intervention or with an alteration of the therapy algorithm .
one patient had a gastric perforation at the gastroesophageal junction during explant of the device following the participant 's decision to discontinue in the study .
table 4 shows the serious adverse events ( saes ) that occurred through 18 months classified according to the clavien - dindo scale .
this analysis demonstrates that 56% of the saes were grade i in severity , 6% were grade ii
( and this patient was not implanted but needed a transfusion due to bleeding from biopsy of a cirrhotic liver ) , 31% were grade iii , and 6% was grade iv .
results of the recharge trial at the 18-month time point provide important context for weighing the benefits and risks of vbloc therapy .
second , vbloc appears to have a favorable safety profile with a low risk of serious complications ( 0.6% of patients had a grade iv complication ) , and nonserious complications were typically mild or moderate sensations of the therapy that were resolved with little to no intervention .
interestingly , weight loss in the sham group was considerably diminished within 6 months of the 12-month endpoint , despite continued blinding of the study past the 12-month visit and ongoing weight management counseling .
these 18-month data were the topic of a meeting of the fda gastroenterology and urology devices panel in june 2014 to consider us regulatory approval of the maestro rechargeable system to deliver vbloc therapy .
the independent panel voted that the benefits of vbloc therapy outweighed the risks , and fda subsequently granted approval to the maestro rechargeable system in january 2015 . given that a large proportion of persons with moderate to severe obesity do not present for traditional bariatric surgical procedures secondary to the concerns for serious complications and permanent alteration of their gastrointestinal anatomy [ 4 , 8 ] , access to additional less - invasive options
while longer - term efficacy data are needed , the continued durability of weight loss with vbloc through 18 months provides additional support that vagal block may be considered an effective alternative to conventional weight loss surgery .
weight loss in the sham group was thought to result from a combination of sham surgery including a sham device , self - monitoring due to daily interaction with the sham device to recharge the battery , and the weight management program .
weight loss in the sham group was more than expected , since participants were not prescribed a diet ( e.g. , portion - controlled meals ) or physical activity , and as such , the primary 12-month objectives of the trial were not met .
sham surgeries in other contexts have also produced large effects [ 911 ] , but these effects would be expected to be transient since no active treatment is being delivered . surprisingly , weight loss in the sham group of the recharge trial was relatively stable between 6 months and 12 months but deteriorated considerably thereafter .
we suspect that this prolonged sham effect may have resulted from enhanced self - monitoring due to the daily interaction with the sham device .
statistical modeling of the 18 month results suggest that unblinding of participants did not have a significant impact on the weight trajectories of either group and that weight regain in the sham group occurred regardless of whether or not participants remained blinded .
this 50% relative increase in the treatment effect of vbloc therapy through 18 months compared to 12 months indicates more substantial efficacy than that previously reported with vbloc compared to a rigorous sham control .
however , nearly 80% of randomized participants in both groups had a visit within 2 months of the 18-month time point and all analyses were conducted on the itt sample using all available longitudinal data , so inference can be made to the entire cohort at 18 months .
this limitation is offset by the continued analysis of the itt cohort rather than a convenience sample as well as sensitivity analyses that concurred with the overall analysis .
finally , all participants were not blinded through 18 months and were unblinded on a rolling basis , making interpretation more difficult .
however , nearly 90% of participants were still blinded through the 15-month visit , at which point much of the weight regain in the sham group had already taken place .
additional analyses also suggest that unblinding did not impact weight regain in the sham group .
follow - up through 18 months of the recharge study showed sustained weight loss with intermittent vagal nerve block but not with a sham surgery and device intervention .
additional long - term data and continued follow - up of the recharge study are needed to further characterize the safety and effectiveness profile of vbloc therapy . | background / objectives . vagal block therapy ( vbloc ) is effective for moderate to severe obesity at one year .
subjects / methods .
the recharge trial is a double - blind , randomized controlled clinical trial of 239 participants with body mass index ( bmi ) of 40 to 45 kg / m or 35 to 40 kg / m with one or more obesity - related conditions .
interventions were implantation of either vbloc or sham devices and weight management counseling .
mixed models assessed percent excess weight loss ( % ewl ) and total weight loss ( % twl ) in intent - to - treat analyses . at 18 months , 142 ( 88% ) vbloc and 64 ( 83% ) sham patients remained enrolled in the study . results .
18-month weight loss was 23% ewl ( 8.8% twl ) for vbloc and 10% ewl ( 3.8% twl ) for sham ( p < 0.0001 ) .
vbloc patients largely maintained 12-month weight loss of 26% ewl ( 9.7% twl ) .
sham regained over 40% of the 17% ewl ( 6.4% twl ) by 18 months .
most weight regain preceded unblinding .
common adverse events of vbloc through 18 months were heartburn / dyspepsia and abdominal pain ; 98% of events were reported as mild or moderate and 79% had resolved . conclusions . weight loss with vbloc
was sustained through 18 months , while sham regained weight between 12 and 18 months .
vbloc is effective with a low rate of serious complications . |
traumatic intracranial hemorrhage ( ich ) , a worldwide health problem , is a major cause of mortality in young adults , and it is associated with high rates of lifelong disabilities among survivors .
in addition to disabilities , survivors of traumatic ich often experience neurocognitive deficits and psychological problems , such as depression , poor decision - making ability , and impulsive aggressive behaviors , leading to poor community , social , and vocational integration .
previous studies have suggested that the detrimental effects of traumatic brain injury ( tbi ) often extend beyond the hemorrhage area .
metabolic changes have also been observed in systemic regions distant from the focal hemorrhagic lesions .
moreover , moderate - to - severe tbi induces several inflammatory signals , which increase proinflammatory cytokine and chemokine release , resulting in monocyte activation and infiltration , glial activation , neuronal and myelin loss , and persistent inflammation .
coronary artery disease ( cad ) , with atherosclerosis being the main pathophysiological mechanism , is the leading cause of morbidity and mortality worldwide .
atherosclerosis , a chronic inflammatory disease of the arterial walls , is characterized by the formation of lipid - laden lesions .
furthermore , lipid deposition , both innate and adaptive inflammation , such as t - cell and macrophage activation , and oxidative stress contribute to atherosclerosis development . among them
, inflammation mechanically alters conventional cad risk factors , such as diabetes mellitus ( dm ) , hyperlipidemia , hypertension , and biological modifications of the arterial walls , which play a major role in atherosclerosis .
although inflammatory mechanisms potentially link traumatic ich and cad , evidence regarding this association is limited . by analyzing taiwan 's national health insurance research database ( nhird )
, we explored the risk of cad in patients with traumatic ich and observed an association between them .
therefore , both patients and clinicians should consider the risk of cad following traumatic ich .
the national health insurance ( nhi ) program , launched by the taiwan government in 1995 , provides comprehensive health care coverage for 99% of the residents in taiwan ( registered in nhird , taiwan ; http://nhird.nhri.org.tw/en/background.html ) .
the national health research institute audits and releases the nhird for use in health service studies .
the nhird comprises reimbursement claims data , including registry of beneficiary , medical record for each insured patient , and medical services .
following regulations implemented by the department of health , the identity of each patient was encrypted for privacy and data security .
for this study , we used a subset of the nhird , including files of inpatient claims and registry of beneficiaries .
this study was exempted from a complete ethical review by the international review board , china medical university , and hospital research ethics committee ( irb permit number : cmuh104-rec2 - 115 ) . from the inpatient claims data
, we identified patients newly diagnosed with traumatic ich ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] 852 and 853 ) from 2000 to 2010 .
the exclusion criteria are outlined as follows : diagnosis of cad ( icd-9-cm 410 - 414 ) at the baseline , aged < 20 years , or having incomplete demographic information .
patients with traumatic ich were frequency - matched to those without traumatic ich and cad at the baseline and by the year of the index date and age ( 5-year intervals ) by using the aforementioned exclusion criteria .
all patients were monitored until cad was diagnosed , loss to follow - up , death , withdrawal from the nhi program , or december 31 , 2011 , whichever occurred first .
each patient was screened at the baseline for preexisting comorbidities , including dm ( icd-9-cm 250 ) , hypertension ( icd-9-cm 401 - 405 ) , hyperlipidemia ( icd-9-cm 272 ) , chronic obstructive pulmonary disease ( copd ; icd-9-cm 490 - 496 ) , chronic kidney disease ( ckd ; icd-9-cm 580 - 589 ) , congestive heart failure ( chf ; icd-9-cm 428 ) , depression ( icd-9-cm 296.2 , 296.3 , 300.4 , and 311 ) , and anxiety ( icd-9-cm 300 ) .
the demographic and clinical characteristics , including sex , age ( 49 , 5064 , and 65 years ) , and baseline comorbidity , of all patients were compared using the test .
we used the student t test for examining the continuous variables ; we plotted the kaplan
meier curve for measuring the cumulative incidence of cad for each cohort and tested the difference by using the log - rank test .
the overall sex- , age- , and comorbidity - specic incidences of cad ( per 1000 person - year ) were calculated for each cohort .
univariate and multivariate cox proportional hazard regression models ( hazard ratios [ hrs ] and 95% confidence intervals [ cis ] ) were used for assessing the risk of cad associated with traumatic ich and then comparing the risk between the 2 cohorts .
the multivariate model was simultaneously adjusted for age , sex , dm , hypertension , hyperlipidemia , copd , ckd , chf , depression , and anxiety .
data were analyzed for evaluating the cad - associated risk factors and the coeffects of cad and traumatic ich .
all analyses were conducted using sas statistical software ( version 9.4 for windows ; sas institute , inc , cary , nc ) .
the national health insurance ( nhi ) program , launched by the taiwan government in 1995 , provides comprehensive health care coverage for 99% of the residents in taiwan ( registered in nhird , taiwan ; http://nhird.nhri.org.tw/en/background.html ) .
the national health research institute audits and releases the nhird for use in health service studies .
the nhird comprises reimbursement claims data , including registry of beneficiary , medical record for each insured patient , and medical services .
following regulations implemented by the department of health , the identity of each patient was encrypted for privacy and data security .
for this study , we used a subset of the nhird , including files of inpatient claims and registry of beneficiaries .
this study was exempted from a complete ethical review by the international review board , china medical university , and hospital research ethics committee ( irb permit number : cmuh104-rec2 - 115 ) .
from the inpatient claims data , we identified patients newly diagnosed with traumatic ich ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] 852 and 853 ) from 2000 to 2010 .
the exclusion criteria are outlined as follows : diagnosis of cad ( icd-9-cm 410 - 414 ) at the baseline , aged < 20 years , or having incomplete demographic information .
patients with traumatic ich were frequency - matched to those without traumatic ich and cad at the baseline and by the year of the index date and age ( 5-year intervals ) by using the aforementioned exclusion criteria .
all patients were monitored until cad was diagnosed , loss to follow - up , death , withdrawal from the nhi program , or december 31 , 2011 , whichever occurred first .
each patient was screened at the baseline for preexisting comorbidities , including dm ( icd-9-cm 250 ) , hypertension ( icd-9-cm 401 - 405 ) , hyperlipidemia ( icd-9-cm 272 ) , chronic obstructive pulmonary disease ( copd ; icd-9-cm 490 - 496 ) , chronic kidney disease ( ckd ; icd-9-cm 580 - 589 ) , congestive heart failure ( chf ; icd-9-cm 428 ) , depression ( icd-9-cm 296.2 , 296.3 , 300.4 , and 311 ) , and anxiety ( icd-9-cm 300 ) .
the demographic and clinical characteristics , including sex , age ( 49 , 5064 , and 65 years ) , and baseline comorbidity , of all patients were compared using the test .
we used the student t test for examining the continuous variables ; we plotted the kaplan
meier curve for measuring the cumulative incidence of cad for each cohort and tested the difference by using the log - rank test .
the overall sex- , age- , and comorbidity - specic incidences of cad ( per 1000 person - year ) were calculated for each cohort .
univariate and multivariate cox proportional hazard regression models ( hazard ratios [ hrs ] and 95% confidence intervals [ cis ] ) were used for assessing the risk of cad associated with traumatic ich and then comparing the risk between the 2 cohorts .
the multivariate model was simultaneously adjusted for age , sex , dm , hypertension , hyperlipidemia , copd , ckd , chf , depression , and anxiety .
data were analyzed for evaluating the cad - associated risk factors and the coeffects of cad and traumatic ich .
all analyses were conducted using sas statistical software ( version 9.4 for windows ; sas institute , inc , cary , nc ) .
our study included 128,997 patients with traumatic ich and 257,994 patients without traumatic ich . among patients with traumatic ich ,
66.3% were males , and 40.1% were aged < 49 years ( table 1 ) .
the mean age of patients with and without traumatic ich was 55.3 19.4 and 54.9 13.8 years , respectively .
furthermore , patients with traumatic ich were more predisposed to the comorbidities , such as dm , hypertension , hyperlipidemia , copd , ckd , chf , depression , and anxiety , compared with those without traumatic ich .
the mean follow - up duration was 4.32 3.41 and 5.41 3.16 years in patients with and without traumatic ich , respectively .
meier analysis results show that the cumulative incidence curves of cad were significantly higher in the traumatic ich cohort than in the comparison cohort ( log - rank test p < 0.001 ) .
the overall incidence of cad in the traumatic ich cohort was 11.4 per 1000 person - years , which was 1.26-fold higher than that in the comparison cohort ( 8.97 per 1000 person - years ) .
after adjustment for age , sex , and comorbidity , the risk of cad was higher in patients with traumatic ich than in those without traumatic ich ( adjusted hr [ ahr ] = 1.16 , 95% ci = 1.131.20 ) .
furthermore , the risk of cad in patients with traumatic ich , but without comorbidities , was significantly higher than that in patients without traumatic ich stratified by sex ( females : ahr = 1.20 , 95% ci = 1.271.41 ; males : ahr = 1.15 , 95% ci = 1.111.19 ) , age ( 49 years : ahr = 1.44 , 95% ci = 1.291.60 ; 5064 years : ahr = 1.07 , 95% ci = 1.001.15 ; > 65 years : ahr = 1.09 , 95% ci = 1.051.13 ) , and comorbidity ( patients without comorbidity : ahr = 1.22 , 95% ci = 1.171.28 ) . demographic characteristics and comorbidity in patient with and without traumatic ich compared with patients without traumatic ich or underlying comorbidities , those with only traumatic ich or comorbidities were associated with a higher risk of cad ( table 3 ) . compared with patients with traumatic ich without comorbidities ,
those with 6 comorbidities had a significantly increased risk of cad ( ahr = 15.9 , 95% ci = 7.9331.7 ) , followed by those with 5 ( ahr = 6.61 , 95% ci = 4.469.80 ) , 4 ( ahr = 5.95 , 95% ci = 5.066.98 ) , 3 ( ahr = 4.16 , 95% ci = 3.794.56 ) , and 2 ( ahr = 2.75 , 95% ci = 2.592.92 ) comorbidities and those with only 1 comorbidity ( ahr = 2.02 , 95% ci = 1.922.12 ) .
joint effects for coronary artery disease between traumatic ich and coronary artery disease - associated risk factor
this large - scale , population - based study indicated that patients with traumatic ich , both men and women , have a significantly higher risk of cad than those without traumatic ich , particularly those without comorbidities ( fig . 1 ,
after adjustment for age , sex , and underlying comorbidities , such as dm , hypertension , and hyperlipidemia , patients with ich were associated with a 16% increased risk of cad ( table 2 ) .
ich is an independent risk factor for cad ; patients with ich have additive effects on the risk of cad when combined with other comorbidities ( table 3 ) , suggesting an association between traumatic ich and cad .
comparison of the cumulative incidence of cad between patients with and without traumatic ich by using the kaplan meier method .
comparison of incidence and hazard ratio of coronary artery disease stratified by sex , age , and comorbidity between with and without traumatic ich patients the nhird is a large - scale , nationwide , population - based database , covering approximately 23 million residents in taiwan and thus permitting several study designs , such as cross - sectional , case - control , retrospective cohort analyses , and family studies . because all patients in the nhi program receive optimal medical care at low costs , loss to follow - up is low , which may provide approximately 20 years of follow - up data of the enrolled population .
advantages of the nhird include its large sample size and lack of selection and participation bias . in this study , specialists diagnosed and coded ( icd-9-cm ) cad and traumatic ich according to the standard diagnosis criteria , including typical symptoms and signs , laboratory data , and imaging findings .
because these data are monitored and strictly evaluated by the bureau of nhi for reimbursement purposes , the diagnoses of cad and traumatic ich are highly reliable .
previous studies have suggested that tbi induces interleukin ( il)-1 and tumor necrosis factor - alpha ( tnf- ) , which triggers an inflammatory cascade and results in an impaired blood
moreover , tbi activates the innate immune system , resulting in the generation of reactive oxygen species ( ros ) , such as superoxide or peroxynitrite , which then cause oxidative damage and long - term neurological deficits .
these inflammatory reactions occur in the central nervous and systemic circulatory system . in atherosclerosis , proinflammatory cytokines , such as il-1 and tnf- , play central roles in the inflammatory cascade .
both in vitro and in vivo investigations suggest that the deposition of cholesterol crystals activates inflammasome , leading to il-1 production , further initiating fatty streaks and promoting local atherosclerotic progression .
tsimikas et al demonstrated that proinflammatory il-1 genotypes were associated with the cad risk and cardiovascular events , which was mediated by oxidized phospholipids and lipoprotein(a ) .
on the basis of the inflammatory nature of atherosclerosis , trials involving anti - inflammatory therapies targeting il-1 and tnf- in cardiovascular disease are ongoing .
moreover , the retained low - density lipoproteins undergo oxidation and modification by ros and then activate endothelial cells and facilitate foam cell formation , which is considered the initial step in the formation of atherosclerotic plaque .
the inflammatory mechanisms linking tbi and atherosclerosis may partly explain the association between traumatic ich and cad in the present study .
our study suggests that the incidence of cad increased in elderly patients and in those with comorbidities , which is consistent with the observation in previous reports .
however , after adjustment for the potential confounding effects by conducting a multivariate cox proportional hazard regression analysis , young age ( < 49 years ) and the absence of comorbidities remained the independent predictors of cad in patients with traumatic ich ( table 2 ) .
younger patients are considered to have a lower incidence of conventional cad risk factors , such as dm , hypertension , and hyperlipidemia .
this is possibly because these patients are more predisposed to traumatic ich alone or the presence and accumulation of complex comorbidities and potential confounding factors reduce the effects of traumatic ich .
we evaluated the coeffects for cad in patients with traumatic ich and cad - related risk factors and observed that although traumatic ich significantly contributes to the cad risk , conventional cad risk factors , such as dm , hypertension , and hyperlipidemia , predominate the development of cad ( table 3 ) .
however , compared with the general population , patients with traumatic ich and dm , hypertension , hyperlipidemia , copd , ckd , and chf exhibited multiplicative risks of developing cad .
these interaction analysis results revealed that the potential effects of traumatic ich on cad must be considered in clinical practice .
there were some limitations in the study : not available personal data of the lifestyles or habits as potential confounding factors for cad in the nhird ( such as body mass index , socioeconomic status , family history , smoking , and alcohol ) ; lack of the individual data of the severity of ich and cad , functional status after traumatic ich , and outcomes with ich and cad ; the medication compliance could not be evaluated by the nhird , leading to the underestimation of the medication effects ; we could not use the additional procedure codes to verify the cad population , although the diagnosis of cad was strictly monitored by certified medical reimbursement specialists in the nhird ; because we used icd-9 codes to evaluate the risk of cad instead of the screening program , therefore these asymptomatic patients might not ask health care until they had the symptoms or sign of cad , leading to the underestimation of the risk of asymptomatic cad in these ich patients ; we could not specify the spectrums of cad ( such as stable and unstable angina and acute myocardial infarction ) in this study ; and the evidence of the methodological quality is usually lower by a retrospective cohort study ( which lacks these necessary adjustments including these possibly unmeasured or unknown confounding factors ) than that by prospective randomized trials . as per our review of relevant literature , this is the first study to analyze data from the nhird and reveal the association between traumatic ich and an increased risk of cad in a large - scale , population - based cohort .
patients with traumatic ich , particularly those with dm , hypertension , and hyperlipidemia , should be educated about the risk of cad . although additional prospective randomized studies are necessary for verifying the effects of traumatic ich on cad , meticulous evaluation and aggressive risk reduction for cad are recommended in patients with traumatic ich . | abstracttraumatic intracranial hemorrhage ( ich ) is prevalent worldwide with long - term consequences , including disabilities .
however , studies on the association of traumatic ich with coronary artery disease ( cad ) are scant
. therefore , this study explored the aforementioned association in a large - scale , population - based cohort.a total of 128,997 patients with newly diagnosed traumatic ich and 257,994 age- and sex - matched patients without traumatic ich from 2000 to 2010 were identified from taiwan 's national health insurance research database .
the kaplan meier method was used for measuring the cumulative incidence of cad in each cohort .
cox proportional regression models were used for evaluating the risk of cad in patients with and without traumatic ich and for comparing the risk between the 2 cohorts.the kaplan meier analysis revealed that the cumulative incidence curves of cad were significantly higher in patients with traumatic ich than in those without ich ( log - rank test , p < 0.001 ) .
after adjustment for age , sex , and comorbidities , patients with traumatic ich were associated with a higher risk of cad compared with those without traumatic ich ( adjusted hazard ratio = 1.16 , 95% confidence interval = 1.131.20 ) .
compared with the general population , patients with traumatic ich and having underlying comorbidities , including diabetes , hypertension , hyperlipidemia , chronic obstructive pulmonary disease , chronic kidney disease , and congestive heart failure , exhibited multiplicative risks of developing cad.this cohort study revealed an increased risk of cad in patients with traumatic ich .
therefore , comprehensive evaluation and aggressive risk reduction for cad are recommended in these patients . |
medication overuse headache ( moh ) is a chronic headache disorder due to overuse of analgesic medicines , taken to treat a primary headache , and remitted by drug discontinuation .
moh has long been considered as a biobehavioural disorder , in which the biological predisposition to develop a chronic headache interacts with an individual behaviour prone to develop a dependence disorder [ 2 , 3 ] .
the molecular basis of moh are largely unknown , though it has been hypothesized that this subtype of headache could be mediated by cognitive impulsivity , and shares some pathophysiological mechanisms with drug addiction , including dysfunction of the frontostriatal system and central pain sensitization induced by drug overuse .
based on this hypothesis , there seems to be a role in moh for bdnf ( brain - derived neurotrophic factor ) , because of its role in central pain sensitization and its relationship with substance - related disorders [ 5 , 6 ] .
bdnf is a member of neurotrophin family , a unique group of polypeptide growth factors that influence differentiation and survival of neurons in the developing nervous system .
bdnf is involved in long - term potentiation ( ltp ) , and therefore in learning and memory mechanisms .
also , a pathogenic role for bdnf has been shown in pain transduction and sensitization , in epilepsy , and in neuropsychiatric disorders .
bdnf gene , mapped to chromosome 11p , encodes for pro - bdnf that is subsequently cleaved in its mature form .
recently , it has been observed that a common 196 g > a single - nucleotide polymorphism ( snp ) of bdnf gene , resulting in a valine at residue 66 to methionine ( val66met ) substitution in pro - bdnf protein , exhibits a functional relevance in that it seems to reduce bdnf activity by altering neurotrophin trafficking .
the val66met variant affects anatomy , cognition and behaviour in adults and leads to memory impairment and susceptibility to obsessive - compulsive , eating , substance abuse and mood disorders [ 6 , 810 ] .
the rationale behind our study derives from two findings , which have been consistently reported in previous researches : ( 1 ) overuse of analgesic drugs is a defining feature of moh and , for this reason , moh has been related to behavioural syndromes associated with substance abuse [ 13 ] , ( 2 ) there is evidence that proneness to use / abuse substances is associated with the allele a of bdnf polymorphism .therefore , we tested the frequency of the val66met genotype in a sample of moh patients , and compared clinical features between individuals homozygous for g allele ( g / g ) and carriers of the allele a ( non - g / g ) .
aim of this study is to confirm our hypothesis that allele a of the examined bdnf polymorphism interfere with moh , seeking for clinical and psychiatric differences between the two groups , mainly in the monthly drug consumption , that is both the clinical hallmark of disease , and the feature closest to abuse disorder .
we would like to emphasize that , although such studies are necessary to get a deeper understanding of the association between moh and bdnf polymorphism , the present study did not intend to ascertain whether the bdnf polymorphism is a risk factor for the development of moh , but only an aggravating factor for subjects that have already developed the disorder .
unrelated consecutive moh patients were enrolled from two italian university headache clinics : the inpatient headache - unit of irccs c. mondino institute of neurology foundation ( pavia ) , and headache outpatient department of university of rome la sapienza , among those admitted with a diagnosis of probable moh , to undergo withdrawal from their overused medications .
although revised criteria were formulated for moh , patients enrolment was started before their statement , thus original ichd - ii version ( i.e. in the presence of headache improvement within 2 months after analgesics withdrawal ) , that is more conservative , was adopted to recruit all patients ; a retrospective analysis will also confirm that all enrolled patients fulfilled the revised criteria .
socio - demographic and clinical data were obtained by anamnesis ; consumption of analgesic drugs per month ( from here , monthly drug number ) , days with headache per month ( from here , monthly headache days ) and days with drug consumption per month ( from here , monthly drug days ) were self reported by patients through a headache diary , received at the time of first visit .
inclusion criteria were : ( a ) patient written informed consent , ( b ) absence of other major medical conditions , ( c ) absence of psychiatric disorders that could sustain a chronic pain condition ( psychotic and/or somatoform disorders ) , ( d ) to have accurately completed the last 3-month pre - withdrawal diary , ( e ) ability to complete the psycho - diagnostic assessment , ( f ) diagnostic confirmation of moh after withdrawal .
presence of categorical psychiatric diagnosis was investigated through four modules of the structured clinical interview for dsm - iv axis i disorders ( scid - i ) , clinician version : mood episodes , mood disorders , anxiety disorders , and eating disorders . moreover ,
a dimensional assessment was made with the beck depression inventory ( bdi ) , a self - reported questionnaire to measure the presence and severity of depressive symptoms . with the patients
written informed consent , 10 cc of peripheral blood were collected , total genomic dna purified , and genotyping for the bdnf val66met - bdnf variant was performed essentially as reported .
all data obtained in the three phases of the study ( socio - demographic and clinical recordings , psychiatric assessment , and genetic determinations ) were merged in a all - comprehensive database by an independent data - manager , which opened the blind and performed the sample size analysis on a preliminary group , to indicate the minimal number of patients needed in the study , and statistical analyses on definitive data . on the base of initial 36 patients ,
a sample size analysis was performed to obtain the required number of subjects to fulfil a desired power of 80% with significance level at 5% . because our primary endpoint was to detect a difference in number of analgesics consumed per month by patients between the two groups ( see introduction ) ,
we divided g / g from non - g / g subjects and calculated the monthly mean of drug consumption : 21 non - g / g subjects consumed meanly 45.57 doses versus 32.93 doses of 15 g / g subjects ( t = 1.887 , p = 0.07 , cohen s d = 0.65 ) .
sample size resulted to be 87 subjects , respectively , 51 for non - g / g and 36 for g / g group .
descriptive statistics to compare bdnf genotypes were performed using parametric or , when appropriate , non - parametric tests .
a stepwise multiple linear regression analysis was employed to identify significant predictors of the monthly drug number in the entire sample .
in addition , to well - know indexes of null hypothesis significance testing , effect size measures were also reported to recognize the value of the degree of association among variables , particularly between bdnf polymorphism group and monthly drug number [ 17 , 18 ] .
effect sizes were calculated with the phi ( ) coefficient [ or , when appropriate , cramr s v ( c ) ] for test , with cohen s d for student s t test , with partial eta squared \documentclass[12pt]{minimal }
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\begin{document}$$ ( \eta_{\text{p}}^{2 } ) $ $ \end{document } for the analysis of variance ( anova ) , and with cohen s f for multiple regression .
unrelated consecutive moh patients were enrolled from two italian university headache clinics : the inpatient headache - unit of irccs c. mondino institute of neurology foundation ( pavia ) , and headache outpatient department of university of rome la sapienza , among those admitted with a diagnosis of probable moh , to undergo withdrawal from their overused medications .
although revised criteria were formulated for moh , patients enrolment was started before their statement , thus original ichd - ii version ( i.e. in the presence of headache improvement within 2 months after analgesics withdrawal ) , that is more conservative , was adopted to recruit all patients ; a retrospective analysis will also confirm that all enrolled patients fulfilled the revised criteria .
socio - demographic and clinical data were obtained by anamnesis ; consumption of analgesic drugs per month ( from here , monthly drug number ) , days with headache per month ( from here , monthly headache days ) and days with drug consumption per month ( from here , monthly drug days ) were self reported by patients through a headache diary , received at the time of first visit .
inclusion criteria were : ( a ) patient written informed consent , ( b ) absence of other major medical conditions , ( c ) absence of psychiatric disorders that could sustain a chronic pain condition ( psychotic and/or somatoform disorders ) , ( d ) to have accurately completed the last 3-month pre - withdrawal diary , ( e ) ability to complete the psycho - diagnostic assessment , ( f ) diagnostic confirmation of moh after withdrawal .
presence of categorical psychiatric diagnosis was investigated through four modules of the structured clinical interview for dsm - iv axis i disorders ( scid - i ) , clinician version : mood episodes , mood disorders , anxiety disorders , and eating disorders . moreover ,
a dimensional assessment was made with the beck depression inventory ( bdi ) , a self - reported questionnaire to measure the presence and severity of depressive symptoms .
with the patients written informed consent , 10 cc of peripheral blood were collected , total genomic dna purified , and genotyping for the bdnf val66met - bdnf variant was performed essentially as reported .
all data obtained in the three phases of the study ( socio - demographic and clinical recordings , psychiatric assessment , and genetic determinations ) were merged in a all - comprehensive database by an independent data - manager , which opened the blind and performed the sample size analysis on a preliminary group , to indicate the minimal number of patients needed in the study , and statistical analyses on definitive data . on the base of initial 36 patients ,
a sample size analysis was performed to obtain the required number of subjects to fulfil a desired power of 80% with significance level at 5% . because our primary endpoint was to detect a difference in number of analgesics consumed per month by patients between the two groups ( see introduction ) ,
we divided g / g from non - g / g subjects and calculated the monthly mean of drug consumption : 21 non - g / g subjects consumed meanly 45.57 doses versus 32.93 doses of 15 g / g subjects ( t = 1.887 , p = 0.07 , cohen s d = 0.65 ) .
sample size resulted to be 87 subjects , respectively , 51 for non - g / g and 36 for g / g group .
descriptive statistics to compare bdnf genotypes were performed using parametric or , when appropriate , non - parametric tests .
a stepwise multiple linear regression analysis was employed to identify significant predictors of the monthly drug number in the entire sample .
in addition , to well - know indexes of null hypothesis significance testing , effect size measures were also reported to recognize the value of the degree of association among variables , particularly between bdnf polymorphism group and monthly drug number [ 17 , 18 ] .
effect sizes were calculated with the phi ( ) coefficient [ or , when appropriate , cramr s v ( c ) ] for test , with cohen s d for student s t test , with partial eta squared \documentclass[12pt]{minimal }
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\begin{document}$$ ( \eta_{\text{p}}^{2 } ) $ $ \end{document } for the analysis of variance ( anova ) , and with cohen s f for multiple regression .
a hundred and seven moh patients ( 84 women and 23 men ; age , mean sd : 46.741 11.36 years ) were enrolled in the study . depending on the type of overused drug ,
patients were divided into four groups : 29 patients ( 27.10% ) consumed exclusively triptans , 18 ( 16.82% ) exclusively non - steroidal antinflammatory drugs ( nsaids ) ; 27 ( 25.23% ) exclusively combination drugs ( combination ) , and 33 ( 30.84% ) associated different types of drugs ( association ) .
none of the patients consumed opioid drugs ; all types of combination drugs consumed by our patients resulted to contain caffeine . by genotyping entire moh population , we identified seven cases of harboured a / a genotype ( 6.54% ) , 53 were g / a ( 49.53% ) , and 47 were g / g ( 43.93% ) .
samples were in hardy - weinberg equilibrium ( = 2.460 , df = 1 , p = 0.12 for the null hypothesis ) .
a preliminary analysis was performed to investigate differences in monthly drug numbers between a / a ( mean sd 54.86 19.05 ) , a /
g ( 49.06 27.19 ) and g / g ( 33.83 14.04 ) group . due to small sample size in a / a group
whitney u test , and the monte carlo method , a bootstrapping technique with a resampling process that produces nonparametric significance estimates .
the three groups differed statistically ( among them ) in number of monthly used drug [ mean rank : a / a , 76.21 ; a / g , 61.43 ; g / g , 42.31 ; h = 13.317 , df = 2 , p < 0.0001 ( 99% confidence interval < 0.00010.001 ) ] .
pairwise comparisons ( significance threshold adjustment with bonferroni s correction , p = 0.0167 ) showed higher values in a / a [ mean rank : a / a , 43.00 ; g / g , 25.19 ; u = 56.000 , z = 2.794 , p2-tailed = 0.004 ( 99% ci 0.0030.006 ) ] and a / g [ mean rank : a / g , 58.82 ; g / g , 41.12 ; u = 804.500 , z = 3.046 , p2-tailed = 0.002 ( 99% ci 0.0010.003 ) ] when compared with g / g patients , but no difference between a / a and a / g [ mean rank : a / a , 37.21 ; a / g , 29.61 ; u = 138.500 , z = 1.082 , p2-tailed = 0.29 ( 99% ci 0.280.30 ) ] . till now obtained results confirmed our decision to consider together carriers of allele a in the non - g / g group : ( 1 ) above - mentioned explorative analysis showed that a / a and a / g groups were not different in terms of monthly drug consumption , ( 2 ) as expected , a little size of a / a subjects was found , and it makes difficult to perform complex , multivariate statistical analysis with an acceptable degree of reproducibility . as mentioned , to perform further statistical analyses , the population was divided into two groups : g / g genotype ( 47 subjects , 43.93% ) , homozygotes for valine ( allele g ) , and non - g / g genotype ( 60 subjects , 56.07% ) , carriers of metionine 66 ( allele a ) , either in heterozygosity or in homozygosity . clinical characteristics and descriptive statistics for the two groups are illustrated in detail in table 1 .
non - g / g showed a higher consumption of monthly drug number ( non - g / g vs. g / g , mean sd : 49.73 26.31 vs. 33.83 14.04 ; t = 4.011 , p < 0.0001 , d = 0.76 ) and longer moh duration ( 6.43 7.78 vs. 3.09 3.66 ; t = 2.944 , p = 0.004 , d = 0.56 ) when compared to g / g patients .
there were no significant differences between g / g and non - g / g patient groups in terms of other clinical and socio - demographic features .
moreover , no significant associations were found among the two groups and current occurrence of psychiatric disorders.table 1table shows descriptive socio - demographic , headache , and psychiatric data for the entire study samplenon - g / g ( n = 60 ; 56.07%)g / g ( n = 47 ; 43.93%)statisticsgender women47 ( 43.93%)37 ( 34.58%) = 0.002p = 0.96 = 0.005 men13 ( 12.15%)10 ( 9.35%)age ( years)47.57 11.4744.94 11.15 t = 1.192p = 0.24d = 0.23education ( years)12.34 4.4012.59 4.09
t = 0.262p = 0.79d = 0.06employment yes38 ( 35.51%)32 ( 29.91%) = 0.263p = 0.61 = 0.050 no22 ( 20.56%)15 ( 14.02% ) age of primary headache onset ( years)16.20 7.4215.32 7.14 t = 0.620p = 0.54d = 0.12 primary headache duration ( years)32.07 11.9630.43 14.28 t = 0.647p = 0.52d = 0.13 age of moh onset ( years)41.53 11.8242.66 12.64 t = 0.475p = 0.64d = 0.09 moh duration ( years)6.43 7.783.09 3.66 t = 2.944p = 0.004d = 0.56 monthly headache days24.82 6.0725.89 5.70 t = 0.935p = 0.35d = 0.18 monthly drug number49.73 26.3133.83 14.04 t = 4.011p < 0.0001d = 0.76 monthly drug days23.63 6.1422.17 6.46 t = 1.195p = 0.24d = 0.23drug type triptans18 ( 16.82%)11 ( 10.28%) = 0.707p = 0.87c = 0.081 nsaids10 ( 9.35%)8 ( 7.48% ) association17 ( 15.89%)16 ( 14.95% ) combination15 ( 14.02%)12 ( 11.21%)current psychiatric diagnosis present45 ( 42.06%)34 ( 31.78%) = 0.096p = 0.76 = 0.030 absent15 ( 14.02%)13 ( 12.15%)current mood diagnosis present27 ( 25.23%)22 ( 20.56%) = 0.035p = 0.85 = 0.018 absent33 ( 30.84%)25 ( 23.36%)current anxiety diagnosis present32 ( 29.91%)24 ( 22.43%) = 0.054p = 0.82 = 0.023 absent28 ( 26.17%)23 ( 21.50%)current eating diagnosis present6 ( 5.61%)2 ( 1.87%) = 1.257p = 0.26* = 0.108 absent54 ( 50.47%)45 ( 42.06%)current double psychiatric diagnosis present18 ( 16.82%)15 ( 14.02%) = 0.045p = 0.83 = 0.021 absent42 ( 39.25%)32 ( 29.91% ) bdi ( score)13.15 8.5312.09 8.73 t = 0.634p = 0.53d = 0.12continuous data are expressed as mean sd , whereas categorical data are frequencies and percentages of total subjects .
differences between non - g / g and g / g patients , with bivariate statistics results , are also illustrated .
bold entries represent the statistical significant differencesmoh medication overuse headache , bdi beck depression inventory , monthly drug days number of days per month in which patient assumed at least one dose of drug to treat headache , drug type triptans ( exclusively one , or more , types of triptan ) , nsaids ( exclusively one , or more , type of non steroidal antinflammatory drugs ) , association ( use of different types of drug in association ) ; combination ( drugs of combination , with more active ingredients ) * fisher s exact test , two - tailed : p = 0.46 table shows descriptive socio - demographic , headache , and psychiatric data for the entire study sample continuous data are expressed as mean sd , whereas categorical data are frequencies and percentages of total subjects .
differences between non - g / g and g / g patients , with bivariate statistics results , are also illustrated .
bold entries represent the statistical significant differences moh medication overuse headache , bdi beck depression inventory , monthly drug days number of days per month in which patient assumed at least one dose of drug to treat headache , drug type triptans ( exclusively one , or more , types of triptan ) , nsaids ( exclusively one , or more , type of non steroidal antinflammatory drugs ) , association ( use of different types of drug in association ) ; combination ( drugs of combination , with more active ingredients ) * fisher s exact test , two - tailed : p = 0.46 a two - way analysis of covariance ( ancova ) was employed to investigate the group effect ( two between - subject factors : genotype , non - g / g vs. g / g ; drug type , triptans vs. nsaids vs. association of drugs vs. drugs of combination ) on monthly drug number ( dependent variable ) ; monthly headache days and monthly drug days were used as covariate in this analysis to reduce the error variance .
the ancova model was highly significant ( f9,97 = 7.149 , p < 0.0001 ) and explained more than one - third of variance ( adjusted r = 0.343 ) . the drug type
effect was not significant ( f3,97 = 0.383 , p = 0.77 , \documentclass[12pt]{minimal }
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drug type was also significant ( f3,97 = 2.776 , p = 0.04 , \documentclass[12pt]{minimal }
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among the covariate , monthly drug days was highly significant ( f1,97 = 16.918 , p < 0.0001 , \documentclass[12pt]{minimal }
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but there was no effect of monthly headache days ( f1,97 = 0.043 , p = 0.84,\documentclass[12pt]{minimal }
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pairwise comparisons [ fisher s least square difference ( lsd ) ; significance threshold adjustment for multiple comparison test with bonferroni s correction ( 0.05/8 ) : p = 0.00625 ] showed no genotype difference in patients who consumed triptans ( p = 0.74 ) and nsaids ( p = 0.16 ) ; on the contrary , when compared with g / g , non - gg patients had a statistically higher consumption of drug both among those that used association of drugs ( p = 0.009 ; this value , however , did not reach the adjusted threshold of significance ) and among consumers of drugs of combination ( p < 0.0001).fig .
1interaction effect of genotype drug type on monthly drug number .
markers ( black circles and squares ) indicate mean , vertical bars denote 0.95 confidence intervals interaction effect of genotype drug type on monthly drug number . markers ( black circles and squares ) indicate mean , vertical bars denote 0.95 confidence intervals as showed in table 2 , in the multiple regression analysis , the val66met bdnf polymorphism ( beta = 0.33 , t = 3.669 , p < 0.0001 , f = 0.134 ) and the bdi score ( beta = 0.19 , t = 2.090 , p < 0.04 , f = 0.037 ) emerged as significant , independent predictors of the monthly drug consumption in moh patients .
no further variables , considered as possible predictors , entered the regression model ( see table 2).table 2results of stepwise multiple regression analysis : bdnf polymorphism ( g / g genotype ) and bdi score ( depressive symptoms ) are the predictors of monthly drug number in moh patientsvariablebetatpbdnf gene0.333.669<0.0001bdi0.192.090<0.04moh duration0.121.2420.22drug type0.080.9170.36gender0.080.9080.37employment0.070.7540.45current psychiatric diagnosis0.050.5320.60age of headache onset0.020.1670.87age0.010.1230.90significant predictors are in boldstepwise model summary : adjusted r = 0.137 , f2,104 = 9.424 , p < 0.0001 , f = 0.181stepwise model change statistics : first step , variable entered = bdnf gene , r change = 0.118 , f1,105 to enter / to remove = 14.030 , p < 0.0001 , f = 0.134 ; second step , variable entered = bdi , r change = 0.036 , f1,104 to enter / to remove = 4.368 , p < 0.04 , f = 0.037moh medication overuse headache ,
bdnf brain - derived neurotrophic factor , bdi beck depression inventory results of stepwise multiple regression analysis : bdnf polymorphism ( g / g genotype ) and bdi score ( depressive symptoms ) are the predictors of monthly drug number in moh patients significant predictors are in bold stepwise model summary : adjusted r = 0.137 , f2,104 = 9.424 , p
< 0.0001 , f = 0.181 stepwise model change statistics : first step , variable entered = bdnf gene , r change = 0.118 , f1,105 to enter / to remove = 14.030 , p < 0.0001 , f = 0.134 ; second step , variable entered = bdi , r change = 0.036 , f1,104 to enter / to remove = 4.368 , p < 0.04 , f = 0.037 moh medication overuse headache ,
bdnf brain - derived neurotrophic factor , bdi beck depression inventory
consistent with our working hypothesis , monthly drug consumption in moh patients resulted to be influenced by the examined bdnf polymorphism ( fig .
2 ) , both if univariate , and both if multiple regression analyses ( to control the association for such possible confounding factors ) are performed .
the relationship between monthly drug consumption and val66met polymorphism was also confirmed by effect size calculation ( medium effect at t test , small to medium effect at regression analysis ) .
the fact that a unique bdnf snp could account for these observed effects is noteworthy , because drug consumption is a multifactorial variable , surely influenced by many other genetic and environmental factors.fig .
g group , the black circles show a / a patients , the grey ones with black ring a / g patients .
vertical bars denote 0.95 confidence intervals monthly drug number in g / g and non - g / g patients .
g group , the black circles show a / a patients , the grey ones with black ring a / g patients .
vertical bars denote 0.95 confidence intervals moh can be considered as a clinical condition at the boundaries between drug addiction and chronic headache .
the hypothetical involvement of bdnf in the pathophysiology of moh was suggested by : ( 1 ) the association between the val66met polymorphism and drug abuse disorders , ( 2 ) the role of bdnf in modulating central and peripheral pain . in this study group ,
non - g / g patients showed a more severe clinical picture , because they had used a higher number of analgesic drugs in the previous 3 months , and had longer disease duration .
showing for the first time a role in moh , our findings confirm that somehow the examined bdnf polymorphism is involved , as an independent factor , in pathophysiology of this headache condition .
although it might sound too approximate , considering the association between met66 pro - bdnf and a reduced bdnf activity , it is tempting to speculate that in moh a lower bdnf activity is a worsening factor .
if moh is regarded as a drug abuse disorder , our data would agree with previous reports of an association between the val66met polymorphism and substance addition .
the proneness to abuse ( i.e. to have an impulse dyscontrol ) , previously reported in non - g / g subjects , would be expressed in moh patients by higher drug consumption . in light of this hypothesis ,
in fact , although the defining factor in moh is the number of days with drug intake , the variability of this value theatrically ranges from 10 to 30 ; on the contrary , a hypothetic upper limit for variability of monthly drug consumption does not exist and patients could be distributed on a wider range that better reflects their proneness to abuse ( and/or headache severity ) .
previous studies have attempted to evaluate bdnf role in headache disorders . in particular , it has been demonstrated an increased level of bdnf in cerebrospinal fluid ( csf ) of patients with a chronic daily headache ( with or without drug overuse ) , in comparison to healthy controls . furthermore , it has been shown a reduced level of platelet bdnf in the interictal phase of episodic migraineurs and cluster headache patients , in comparison to healthy controls .
more recently , a bdnf elevation in csf was demonstrated both in chronic migraine and in fibromyalgic patients .
these three studies were much different from our research for two reasons : the use of diverse classification criteria in the first study ; in general , the different study designs , with the lack of relations among bdnf levels , clinical markers of disease severity , and the patients bdnf genotype . however , these findings showed the involvement in chronic pain pathophysiology of bdnf up regulation , and might support indirectly our hypothesis that in moh patients a bdnf hypo - activity , related to val66met polymorphism , does not influence pain severity , but impulsive behaviour . to the best of our knowledge ,
pending specific studies on this topic , only speculations could be made about the chance that the higher drug intake is a consequence of putative bdnf - mediated higher pain sensitivity , or not .
however , the pain modulation is linked to bdnf release and up - regulation [ 23 , 24 ] , whereas val66met polymorphism is related with a reduction of bdnf activity .
these observations , though indirectly , might lend support the idea that bdnf influence on moh is played in terms of impulsivity modulation , as in other drug abuse disorder , and not pain hypersensitivity . moreover , a recent paper analysed influence of val66met polymorphism on episodic migraine .
examined polymorphism resulted neither associated to development of migraine ( comparing polymorphism frequency in a population of episodic migraineurs with a healthy control group ) , nor associated with differences in terms of clinical characteristics ( within the only migraine group ) .
consistent with these data , because val66met polymorphism seems to be unable to influence development and clinical features of migraine , we suppose that observed worsen effect of polymorphism on moh is likely driven by the a allele related proneness to abuse , and , in turn , that the monthly drug consumption in moh patients is not necessarily the expression of headache severity .
as reported above , we have also recorded the type of drug consumed by each patient and they were segregated in four groups : triptans , nsaids , association of different type of drugs and drugs of combination .
it is noteworthy that differences between g / g and non - g / g emerged only in the fourth group ( drugs of combination , all containing caffeine ) , and a trend was also found for the association of drugs . if one consider that non - g / g subjects are supposed to have a bdnf hypoactivity , and that bdnf is a kind of biological reward for presynaptic neurons , our observations could mean that the lack of reward induces non - g / g subjects in the continue search of different type of drugs , or in caffeine seeking ( for its psychotropic effect ) .
none of the patients reported to use drugs containing opioids ; therefore , we can not confirm our hypothesis with this class of drugs .
severity of depressive symptoms ( as measured by bdi score ) was found as another independent predictor of drug consumption in moh patients .
the role of depression in headaches has been previously reported and discussed elsewhere [ 26 , 27 ] .
although a relation between depression and val66met polymorphism was frequently found , in this study the g / g and non - g / g groups neither differ statistically for the severity of depressive symptoms , nor were they associated significantly with a psychiatric disorder .
maybe these associations are covered in our population by already described relationship between moh and psychiatric comorbidity that could not be further increasable by examined polymorphism . moreover , observed lack of association suggests that this bdnf polymorphism and depression might have an effect on moh monthly drug consumption through different and unrelated mechanisms . in summary ,
our results showed an influence of bdnf polymorphism val66met on drug consumption in moh patients .
the polymorphism is likely not related to severer forms of headache , but with a bdnf hypoactivity , in turn related to substance abuse behaviour , consistent with observed higher caffeine seeking .
therefore , aggravating effect of examined polymorphism in moh patients could be driven by its proneness to develop an impulsivity dyscontrol , the cornerstone of any substance abuse disorder .
in other words , we suppose that moh is not only a very severe headache , but also a complicated bio - behavioural disorder , not dissimilar to a kind of addictive disorder , preferentially developed by some migraineurs , aggravated by conditions unrelated to previous headache clinical features ; however , related to behavioural impulsivity dyscontrol , as other substance abuse disorders .
moreover , we would like to highlight that analysis of snps within functionally related genes appears as a good tool in headache research to identify mechanisms potentially involved in disease pathogenesis . | medication overuse headache ( moh ) can be considered a clinical condition at the boundaries between drug addiction and chronic pain disorder .
the common 196 g > a single - nucleotide polymorphism of bdnf gene , resulting in a valine 66 to methionine ( val66met ) , is related with behaviour disorders and substance abuse . with the aim of identifying a worsening factor in moh , rather than the detection of a specific risk factor for the development of the disease , we investigated whether the presence of a functional bdnf polymorphism might determine clinical differences within a group of 90 moh patients , particularly in monthly drug consumption , that is the hallmark of disease . directly comparing moh patients homozygous for g allele ( g / g ) with carriers of a allele ( non - g / g ) ,
we have observed 47 g / g genotypes and 60 non - g / g genotypes .
non - g / g had a higher consumption of monthly drug number ( cohen s d = 0.76 ) than g / g patients . at multiple regression analysis ,
the val66met bdnf polymorphism emerged as a significant independent predictor of analgesic drug consumption ( beta = 0.33 , cohen s f2 = 0.134 ) .
these findings showed an influence of examined bdnf polymorphism in the moh clinical features , supporting the idea that moh is a substance abuse disorder . |
this performance improvement study used a one - group pre- and postintervention design at an academic 150-bed children 's hospital from february 2006 to january 2008 .
the pediatric nursing staff ( see supplemental table 1 for demographics , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-0031/dc1 ) was required to complete a three - part module ( view at http://rushakoff.com/rushakoff/rushakoff2 ) .
section one provides information regarding types of diabetes , insulin pharmacokinetics , physiological insulin dosing , calculating and administering a diluted insulin dose , recognition and management of hypoglycemia , and insulin pump therapy and explains our institution 's two - rn independent check process for high - alert medications ( two rns independently determine insulin dose from physician order ) .
section two provides information regarding use of the pediatric subcutaneous insulin order set , including timing of injections and glucose monitoring .
section three consists of 15 questions related to interpreting insulin orders , determining the correct insulin dose , and identifying nursing actions based on the blood glucose and carbohydrate amount .
for each question , if answered incorrectly , the nurse was instructed why the selected answer was incorrect and was required to try again . this analytic sample consisted of all pediatric patients during the study periods with a diagnosis of diabetes or hyperglycemia .
all eligible charts were reviewed by two members of the study team ( m.m.s . and c.r.o .
) to identify the number of possible and actual insulin - related medication errors . using a comprehensive audit tool ( supplemental table 2 ) created by the research team ,
retrospective chart audits were conducted for a period of 6 months before implementing the module and 26 months postimplementation .
opportunity for error was defined as each time the blood glucose should have been checked per physician order or the hypoglycemia protocol .
an error was defined as any dose of insulin given or omitted that deviated from the physician order ( omission of an insulin dose , wrong insulin dose or type , wrong administration time , blood glucose not checked per order or hypoglycemia protocol , and blood glucose not documented in the record ) .
the pediatric nursing staff ( see supplemental table 1 for demographics , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-0031/dc1 ) was required to complete a three - part module ( view at http://rushakoff.com/rushakoff/rushakoff2 ) .
section one provides information regarding types of diabetes , insulin pharmacokinetics , physiological insulin dosing , calculating and administering a diluted insulin dose , recognition and management of hypoglycemia , and insulin pump therapy and explains our institution 's two - rn independent check process for high - alert medications ( two rns independently determine insulin dose from physician order ) .
section two provides information regarding use of the pediatric subcutaneous insulin order set , including timing of injections and glucose monitoring .
section three consists of 15 questions related to interpreting insulin orders , determining the correct insulin dose , and identifying nursing actions based on the blood glucose and carbohydrate amount .
for each question , if answered incorrectly , the nurse was instructed why the selected answer was incorrect and was required to try again .
this analytic sample consisted of all pediatric patients during the study periods with a diagnosis of diabetes or hyperglycemia .
all eligible charts were reviewed by two members of the study team ( m.m.s . and c.r.o .
using a comprehensive audit tool ( supplemental table 2 ) created by the research team , retrospective chart audits were conducted for a period of 6 months before implementing the module and 26 months postimplementation .
opportunity for error was defined as each time the blood glucose should have been checked per physician order or the hypoglycemia protocol .
an error was defined as any dose of insulin given or omitted that deviated from the physician order ( omission of an insulin dose , wrong insulin dose or type , wrong administration time , blood glucose not checked per order or hypoglycemia protocol , and blood glucose not documented in the record ) .
patient demographics and details of insulin errors are in table 1 . a total of 100% of the children 's hospital 's registered nursing staff ( n = 283 ) completed the educational module . before intervention
the mean number of errors per patient in this sample was 5.20 . in the postintervention sample ,
the mean number of errors per patient in this sample was 0.86 ( p = 0.003 ) .
the number of patients experiencing insulin - related errors during their hospitalization decreased from 21 out of 24 ( 87.5% ) in the preintervention phase to 5 out of 22 ( 22.7% ) postintervention .
the overall error rate decreased from 14.8% in the preintervention phase to 1.7% in the postintervention phase ( p < 0.001 ) .
study demographics and insulin errors per patient data are means sd or n ( % ) unless otherwise indicated .
glucose may have been found in laboratory data , but not documented as mandated per nursing policy . percentage based on total possible errors .
these findings demonstrate that an interactive online nursing educational module can be an effective strategy for reducing insulin administration errors in hospitalized pediatric patients .
multiple studies have found deficits in nurses ' knowledge regarding diabetes ( 911 ) , suggesting that additional education about diabetes is needed in order for nurses to provide safe and effective care to these patients .
consistent with phillips ' ( 12 ) recommendation that educational modules provide frequent feedback to learners , our module used a self - paced technological medium that provided active learning , problem solving , and immediate feedback to the nurses .
strategies that enhanced implementation included engaging hospital administration support from the beginning of the study ; recruiting nursing unit champions who took ownership of the performance improvement initiative at the unit level and acted as an educational resource for the nursing staff ; and requiring that all nurses view the module .
study limitations include limited generalizability due to a small convenience sample of pediatric nurses at a single academic medical center 's children 's hospital .
second , although the chart reviewers were not involved in the care of these patients , and therefore were blinded to the medication administration process and documentation , the analysis was done by the same team that collected the data , leaving the possibility of investigator bias .
however , the magnitude of the difference between the pre- and postintervention findings makes it likely that a real reduction in errors did in fact occur .
finally , using a retrospective chart audit to detect errors was also a limitation , since it relies on the accuracy and adequacy of nursing documentation . in conclusion , this study presents a first attempt to evaluate an effective and feasible method of educating a large number of nurses on the correct implementation of physiologic insulin dosing for hospitalized pediatric patients .
more research is needed to validate these findings , as well as to determine how to sustain and disseminate successful educational programs . | objectiveto determine the effectiveness of an online module for reducing insulin administration errors by nurses caring for hospitalized pediatric patients.research design and methodspediatric nursing staff completed a mandatory online educational module teaching insulin pharmacokinetics and the insulin order form , using diluted insulin and finishing with 15 interactive cases . a chart audit to determine all possible insulin errors of patients receiving insulin
was done before and 26 months after the educational module.resultsall of the medical center 's 283 pediatric nurses successfully completed the educational module .
a total of 24 charts were audited in the preintervention phase and 22 in the postintervention phase .
the preintervention insulin error rate was 14.8% , reduced to 1.7% ( p < 0.001 ) postintervention .
improvement occurred in correct insulin dosing and type , timing of administration , and timely blood glucose monitoring and documentation.conclusionsan interactive online educational module can be an effective strategy for reducing pediatric nurses ' insulin administration errors . |
thiamine deficiency is common in parts of southeast asia . in some settings , as few as 1 in 30 breastfeeding mothers consumes the recommended daily allowance of thiamine .
this is unsurprising , as the per capita daily thiamine available in the cambodian food supply is estimated to provide only half of the recommended daily allowance for breastfeeding women .
postpartum traditions in parts of southeast asia , including consumption of glutinous white rice , dietary intake restriction , betel nut chewing , and consumption of thiaminase - containing food products such as fermented fish paste , also exacerbate low thiamine status in mother - infant pairs .
unlike many micronutrients , the thiamine content of breast milk is negatively affected by maternal depletion , placing infants at a greater risk for deficiency .
suboptimal levels of this essential micronutrient result in infantile beriberi , a disease characterized by respiratory distress and heart failure .
infantile beriberi was responsible for 40% of infant deaths during early settlement of the maela refugee camp on the thai - burmese border .
heart failure associated with infantile beriberi is still thought to account for death in up to 3% of cambodian children before age five . while thiamine deficiency is widely prevalent , fulminant infantile beriberi is far less common . in a study of cardiac function among thiamine - deficient cambodian children ,
moreover , children with signs and symptoms of beriberi ( eg , tachypnea , tachycardia , vomiting , lethargy , and hepatomegaly ) exhibited moderately low thiamine concentrations comparable to those of healthy cambodian controls . accurately distinguishing children with true infantile beriberi from those children with other illnesses
what causes illness in children who have signs of beriberi and moderate thiamine deficiency without any evidence of cardiac dysfunction ?
previous work done by porter et al and keating et al revealed a positive correlation between respiratory rate and the extent to which tiger balm was applied to infants ( unpublished observations ; r = .41 , p < .01 ) .
tiger balm is a petroleum - based ointment that cambodian mothers commonly apply to infants with pain or colic .
there are various formulations of tiger balm used among southeast asian populations , but camphor is one of the main active ingredients across all brands .
camphor activates transient receptor potential vanilloid ( trpv ) and transient receptor potential ankyrin ( trpa ) channels , super families of nonselective ion channels that are involved in sensory transduction .
trpv channels are expressed in the medial preoptic nucleus of the hypothalamus and may be important for hypothalamic thermo - sensitivity .
thus , we hypothesized that camphor from tiger balm could alter temperature regulation proteins in nuclei of the hypothalamus and stimulate tachypnea as an alternative temperature - regulating behavior , separate from any influence of thiamine deficiency .
therefore , the objective of this secondary analysis investigation was to quantify concentrations of camphor in blood of tachypneic infants and healthy controls in order to determine if symptoms in thiamine - deficient infants without echocardiographic abnormalities could possibly be associated with camphor toxicity from tiger balm .
in the original study , infants aged 2 weeks to 12 months participated in an observational , case - control study at the svay chrum health clinic in the mesang district of prey veng province , cambodia .
we enrolled 20 sick infants meeting clinical criteria for beriberi diagnosis and 42 healthy control infants from the surrounding village between july 10 , 2012 , and august 3 , 2012 .
all mothers gave informed consent for their infants participation in the study , and a survey was given to mothers of all enrolled infants assessing dietary and environmental exposures , including tiger balm use , as described previously . in the present study , we assayed residual frozen whole - blood samples from 9 tachypneic infants meeting clinical criteria for beriberi and 10 healthy control infants , all without either an abnormal echocardiogram or a profoundly low ( < 30 nmol / l ) whole blood thiamine diphosphate ( tdp ) concentration .
median whole blood tdp concentrations were 61.3 nmol / l ( range = 41.3 - 114.0 nmol / l ) in tachypneic infants and 46.6 nmol / l ( range = 38.0 - 59.4
nmol / l ) in healthy control infants ( p = .08 ; table 2 ) .
camphor analysis of whole blood samples was performed using an established laboratory method ( nms labs , willow grove , pa ) .
briefly , after extraction with chloroform , aliquots were analyzed using capillary gas chromatography with a flame - ionization detector .
sample concentrations were determined by comparing the ratio of the area under the peak of camphor with an internal standard and calculating by linear regression against the standard curve .
camphor was linear from 0.02 to 0.50 g / ml , and the standard curve was calibrated at the following concentrations : 0.02 , 0.05 , 0.10 , 0.20 , 0.30 , and 0.50 g / ml . data were recorded as median ( range ) or as percentages and analyzed using jmp software ( sas institute inc , cary , nc ) .
the mayo clinic institutional review board and the cambodian national ethics committee for health research granted ethics approval for the original study , and the mayo clinic institutional review board approved the additional camphor analysis ( no .
detailed information regarding data collection , analysis , interpretation , and ethics approval is available on request from the corresponding author .
basic descriptive data and statistics of enrolled tachypneic and healthy control infants are presented in table 1 .
however , there were significant differences observed by age ( p = .001 ) and by number of people over age 10 who share meals in the household ( p = .01 ) .
tiger balm use among enrolled infants in both study groups was common ( table 2 ) .
eight ( 88.9% ) mothers of tachypneic infants and 7 ( 70% ) mothers of healthy infants reported presently using tiger balm on their child ( p = .36 ) .
six mothers of tachypneic infants and 7 mothers of healthy infants reported applying tiger balm daily on their infant . among infants on whom
tiger balm was applied daily , the median number of applications per day was 1.5 ( range = 1 - 4 applications per day ) for tachypneic infants and 1 ( range = 1 - 2 applications per day ) for healthy control infants ( p = .41 ) .
no case subject and no healthy control subject had any detectable camphor in the blood .
children of the mesang district of prey veng province , cambodia , vary widely in their thiamine status , from normal blood concentrations to severe thiamine deficiency , although clinical illness compatible with beriberi diagnosis correlates poorly with thiamine level .
we analyzed blood samples in tachypneic and healthy control infants for camphor concentrations , testing the hypothesis that camphor from tiger balm use could stimulate tachypnea in cambodian children .
failure to detect even trace amounts of camphor in blood samples suggests camphor toxicity is unrelated to symptomatic clinical illness in these children .
it is conceivable that camphor elicits effects at the tissue level without accumulating in the blood .
this explanation is unlikely , however , due to the lipophilic nature and high volume of distribution of camphor .
mothers of ill children may be more prone to use tiger balm on their infants in response to irritability or other symptoms associated with their illness , potentially explaining the correlation with respiratory rate .
however , other ingredients are included in tiger balm formulations with the potential to cause illness .
high doses of salicylates , for instance , are known to cause metabolic acidosis , which could lead to a compensatory increase in respiration .
if any significant amount of salicylates was present , however , camphor would be expected to be observed in the blood , as well .
nevertheless , a causal relationship between other components of tiger balm and illness in thiamine - deficient cambodian infants can not be completely ruled out .
camphorated products are commonly sold in cambodian markets and are used extensively as a treatment of colic and abdominal discomfort in infants by lay caregivers , and among all ages for various other maladies , including headache , dizziness , and insect bites .
the safety of camphorated products being sold over - the - counter is actively debated , due to toxicity risks .
camphor toxicity is reported in cases of ingestion , inhalation , and prolonged skin contact , with symptoms ranging from mild oropharyngeal , skin , and respiratory irritation to seizures , coma , or death .
seizures , a cardinal symptom of toxicity , are often the only manifestation of camphor intoxication .
onset of symptoms depends on route of exposure : toxicity due to ingestion often manifests within 5 to 90 minutes , whereas in the relatively few cases of dermal intoxication , symptoms appeared within 4 hours to 3 weeks . symptoms generally resolve within 24 hours but may persist for longer periods , especially in cases of dermal intoxication .
toxicity is reached at serum concentrations of 0.3 to 0.4 g / ml ; concentrations of 1.7 g / ml can lead to coma or death .
while ingestion is the most common means of toxicity , repeated or prolonged dermal exposures may also result in symptomatic plasma concentrations : one report revealed 0.45 g / ml of camphor 17 hours after repeated applications . among reports of asymptomatic camphor ingestion , plasma concentrations ranged from undetectable to 0.015
peak plasma concentration after dermal exposure to camphor patches was reached at 4 hours after application , corresponding with onset of symptoms in dermal intoxication .
camphor from dermal patches was undetectable in blood plasma within 8 to 12 hours after exposure , consistent with normal time of symptom dissolution .
important limitations of this retrospective investigation include imprecise and variable dosage of tiger balm among infants and failure to record time from last exposure to initial blood draw .
however , because no camphor was detected in blood drawn from concurrently symptomatic infants , it is unlikely that their symptoms were due to any significant degree of camphor toxicity .
stability of camphor in frozen blood has also not been evaluated , a potential concern for our investigation , as samples were stored for upwards of 30 months before analysis . however , camphor is stable at room temperature for 5 years .
infant ages also varied between study groups , although all cases were less than 5 months old , the age range during which beriberi typically presents .
infantile beriberi can be a devastating disease , and yet it has a simple , actionable solution : thiamine supplementation via direct administration , food fortification , or agricultural intervention .
reports of infantile beriberi are relatively limited to southeast asia where polished rice is a staple food ; even in sub - saharan africa where up to a third of disability - adjusted life years have been attributed to malnutrition , beriberi is rarely observed , likely because staples contain even small amounts of thiamine .
even though not all thiamine - deficient children develop infantile beriberi , this childhood disease is never seen in populations with adequate maternal thiamine intake .
therefore , while there is still an important scientific question as to the pathophysiology behind the presentation of beriberi , there is unequivocal evidence that adequate thiamine status is required to prevent infantile beriberi .
future work among thiamine - deficient populations should account for both the urgency of a curable disease and the need to understand progression from asymptomatic thiamine deficiency to fulminant beriberi for early , accurate diagnosis and prevention of disease .
public health initiatives on the local , national , and international levels will be especially important in early relief efforts , although a sustainable solution will likely require collaborations between government , health professionals , researchers , and public health workers .
future investigations of tachypneic infants in regions with endemic thiamine deficiency could benefit from exploration of additional vitamin or mineral statuses .
thiamine is important for several metabolic processes and interacts with many other intrinsic and extrinsic factors , implying a need to understand more about the overall nutritional status of tachypneic children in cambodia .
other avenues of future investigation include dietary interventions to curb thiamine deficiency , such as was demonstrated recently by whitfield et al through fortification of fish sauce used in cambodian households .
furthermore , although this study and others failed to correlate environmental exposures with illness , the possibility of toxicity from environmental contaminants , including tiger balm , remains to be excluded .
crj : contributed to conception and design ; contributed to analysis and interpretation ; drafted manuscript ; critically revised manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy .
sgp : contributed to conception and design ; contributed to acquisition and interpretation ; critically revised manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy .
dc : contributed to conception and design ; contributed to acquisition and interpretation ; critically revised manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy .
kcw : contributed to analysis and interpretation ; critically revised manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy .
km : contributed to conception and design ; contributed to acquisition and interpretation ; critically revised manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy .
mdt : contributed to conception and design ; contributed to analysis and interpretation ; critically revised manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy .
prf : contributed to conception and design ; contributed to analysis and interpretation ; drafted manuscript ; gave final approval ; agrees to be accountable for all aspects of work ensuring integrity and accuracy . | thiamine deficiency and beriberi are prevalent in cambodia , although most infants with nonspecific clinical symptoms of beriberi , including tachypnea , lack echocardiographic evidence diagnostic of the disease .
camphor activates transient receptor potential vanilloid 3 ( trpv3 ) , a nonselective ion channel expressed in the medial preoptic nucleus of the hypothalamus and thought to be important for thermo - sensitivity .
because camphorated ointments are used commonly among cambodian infants , we hypothesized that topical camphor modulates thermoregulatory behaviors , causing beriberi - simulating tachypnea , separate from any influence of thiamine deficiency .
we assessed 9 tachypneic and 10 healthy infants for tiger balm use and for presence of camphor in whole blood .
however , no camphor was found in blood from any infants , indicating that camphor is unrelated to tachypneic illness in cambodian infants . |
a 48-year - old man was incidentally detected as having a well - defined , heterogeneous enhancing soft tissue density lesion in the bilateral retroperitoneum .
the lesions were found during ct while the patient was undergoing evaluation for a gallbladder polyp that was incidentally detected on ultrasonography .
the ct showed bilateral masses in the anatomical position of the adrenal glands with densities indicating adipose tissue .
the maximum diameter was 8.0 cm for the left mass and 1.8 cm for the right mass .
1 ) . he had a 4-year history of hypertension , for which he was receiving antihypertensive medication .
he had no specific symptoms and the results of a physical examination were also nonspecific .
the biochemistry profile showed an elevated level of plasma epinephrine of 67.8 pg / ml ( range , 1 - 20 pg / ml ) , norepinephrine of 340.8 pg / ml ( range , 15 - 80 pg / ml ) , and acth of 158 pg / ml ( range , 10 - 90 pg / ml ) , and urinary 17-ks of 48.5 mg / day ( range , 8 - 22 mg / day ) and 17-ohcs of 55 mg / day ( range , 5 - 23 mg / day ) .
plasma cortisol , renin , aldosterone , and 24-hour urinary cortisol , free cortisol , metanephrine , vanillylmandelic acid , and homovanillic acid were all within normal limits . in the abdominal mri ,
the left tumor measured 8.2 cm and the right tumor measured 2.0 cm in maximum diameter , had high signal intensity on both t1- and t2-weighted images , and low signal intensity on t1 fat - suppressed images and chemical - shift images ( fig .
a differential diagnosis for malignant tumors was necessary , so the patient was subjected to transperitoneal laparoscopic left adrenalectomy .
on macroscopic examination of the surgically removed specimen , the tumor was measured as 9.14.9 cm , weighed 128 g , and was yellowish - brown in color .
microscopic examination revealed that the tumor was composed of mature fat mixed with hematopoietic elements including megakaryocytes ( fig .
the adrenal gland is the most common site , but myelolipomas are also rarely present in extra - adrenal sites , including the pelvis , mediastinum , retroperitoneum , and paravertebral region , as an isolated soft tissue mass .
adrenal myelolipoma , affecting both sexes in similar rates , usually presents late in adult life , and most cases are diagnosed during the fourth to seventh decade .
the association of myelolipoma with obesity , hypertension , chronic disease , and malignancies has been described .
adrenal myelolipomas are in the majority of cases unilateral and often less than 4 cm in diameter .
the term giant myelolipoma is preferred when the size exceeds 8 cm . in the literature ,
the largest adrenal myelolipoma ( diameter 31 cm , weight 6.0 kg ) was described by akamatsu and colleagues . to our knowledge ,
10 cases of adrenal myelolipoma were reported in korea . however , these cases were all unilateral .
adrenal myelolipoma , which is usually asymptomatic , is generally diagnosed during surgical interventions and imaging procedures performed for other purposes .
our patient also performed ct for evaluation of a gallbladder polyp that was incidentally detected on ultrasonography . in symptomatic cases ,
the most common symptom is nonspecific abdominal pain , probably due to hemorrhage , tumor necrosis , or mechanical compression of adjacent organs from large tumor bulk .
adrenal myelolipomas are generally hormonally inactive , although there are case reports of their association with overproduction of adrenal hormones .
they have been associated with overproduction of dehydroepiandrosterone - sulphate ( dheas ) , congenital adrenal hyperplasia caused by 21-hydroxylase deficiency , congenital adrenal 17 -hydroxylase deficiency , cushing disease , conn 's syndrome , adrenal insufficiency , and pheochromocytoma .
some of these tumors coexist with adrenal adenomas , which may in part account for the hormonal activity . among them , congenital adrenal hyperplasia and cushing syndrome appear to be the two most common endocrine disorders described . in our case ,
the levels of plasma epinephrine , norepinephrine , and acth were elevated , but there was no evidence of a functioning pheochromocytoma in the mibg scan or other hormone - producing tumors in the histopathology .
the pathogenesis of myelolipoma is uncertain , with the prevalent hypothesis suggesting that they arise from the zona fasciculate of the adrenal cortex from metaplasia of undifferentiated stromal cells .
more specifically , they are assumed to arise from metaplasia of either previously uncommitted adrenal cortical mesenchymal cells or of groups of choristomatous hematopoietic stem cells that transmigrate during intrauterine life to the developing adrenal gland .
other theories , less favored , propose embolism of bone marrow cells and development from intra - adrenal embryonic rests of bone marrow .
diagnosis of myelolipoma is based on imaging , with ultrasonography , ct , and mri being effective in more than 90% of cases .
the classic myelolipoma is radiolucent on plain films and avascular on angiography . on ultrasonography ,
a myelolipoma is hyperechoic if it consists mostly of fat and hypoechoic if it is composed primarily of myeloid cells .
ct is the most sensitive imaging modality , and the appearance of myelolipoma depends on its histological composition .
myelolipoma often has a discrete capsule and appears as well - delineated heterogeneous masses with regions of less than -30 hounsfield units that correspond to low - density mature fat . in mri , fat tissue has high signal intensity in both t1 and t2 images , whereas myeloid tissue has low signal intensity in t1 and moderate signal intensity in t2 images .
differential diagnosis includes other fat - containing adrenal masses such as teratoma , lipoma , and liposarcoma , which are less common , and even rarely angiomyolipoma , mass - forming extramedullary hematopoiesis , and adenoma . in case
a diagnosis has not been established , fine needle aspiration can be helpful to definitively rule out malignancy .
previously , most patients with myelolipoma underwent surgical resection for a suspected malignant neoplasm . nowadays , with the frequent detection of these myelolipomas incidentally
, the treatment has been a matter of much debate . when the tumor is 4 cm or smaller and asymptomatic , follow - up with ct
if symptoms occur , surgery should be carried out promptly , especially for a large myelolipoma , because spontaneous rupture of the lesion with hemorrhage is possible .
previously , a tumor size exceeding 5 to 6 cm was thought to be a contraindication for the laparoscopic approach .
but several recent studies have shown that the laparoscopic approach is technically feasible , safe , and comparable with the open approach in these patients . in the case of bilateral adrenal myelolipomas ,
a staged tumor removal is preferable , removing the larger one and continuing to observe the contralateral myelolipoma as long as possible in an effort to avoid adrenal insufficiency and a lifetime of steroid replacement .
our patient had bilateral adrenal myelolipomas in which the left tumor was larger than the right , measuring 8.2 cm vs. 2.0 cm in maximum diameter on the mri .
however , if there were bilateral , giant , symptomatic adrenal myelolipomas in a patient with congenital adrenal hyperplasia , bilateral surgical resection should be done . | adrenal myelolipoma is a rare , nonfunctional benign tumor that is composed of mature adipose tissue and hematopoietic elements . in the past
, these tumors were accidentally discovered at autopsy .
today , they are found much more frequently and incidentally , mainly because of the widespread use of noninvasive imaging with ultrasonography , computed tomography , and magnetic resonance imaging .
most lesions are asymptomatic , small , and unilateral , but a number of bilateral tumors have been reported .
we report here on a case of a 48-year - old man with incidentally found myelolipomas of both adrenal glands .
in particular , a giant myelolipoma of the left adrenal gland was treated by transperitoneal laparoscopic adrenalectomy . |
. it might be easy to simply think about adipocytes as those " pesky " cells that conspire against us to fill with fat as we age to make us look differently . upon a closer look at adipocyte cell lineage origins , physiology , bodily repository locations ( depots ) and also at the adipocyte 's ability to perform a diverse set of metabolic functions other than just simply to " pad " our bodies ( especially in the wrong places ) , it might be surprising to learn that adipocytes have profound impacts on a variety of major bodily systems beyond just lipid storage 1 - 3 . consequently , a focus of this issue of the journal of genomics was to shed light on adipocytes , bring adipocyte biology into the molecular level and to identify gaps in contemporary understanding of various aspects of adipocyte biology -- especially with respect to adipogenesis ( genomics , transcriptomics and metabolomics ) 4 . by doing so ,
we hope to generate enthusiasm in this research area and to ( perhaps ) recruit more people into the arena of adipocytes .
one area that was presented in numerous papers of this issue as segmented informative bits 5 - 8 was the potential for cells found in adipose depots to become other cells , so the " stem cell(s ) " of adipose tissue is / are briefly discussed by this paper .
mesenchymal ( and perhaps other germ layer - origin ) stem cells 9 - 13 , adipofibroblasts 14 , 15 , stromal vascular cells 6 , preadipocytes 6 , 16 , 17 , satellite cells 10,11 and ( even though considered post - mitotic ) 18 mature adipocytes 19 - 39 all provide cells that can assimilate lipid and display lipid metabolism 17 , 40 , 41 . cells such as bone - derived cells not originally displaying the adipocyte phenotype might be induced to become an adipocyte 18 , 34 , 42 , 43 and numerous maladies such as lipotrophic muscular atrophy ( a good model is japanese black cattle ) 44 and steatosis 2,17 have been described whereby original tissue was replaced by adipose - like cells perhaps regulated via intercellular signals / cross talk between muscle and adipose tissue / cells 45 .
this results in cells losing their original function while maintaining original tissue size and structure 46 . embryonic development . in a similar manner to the development of skeletal muscle in utero
, it seems likely that embryonic adipogenesis may involve numerous populations of stem cells 13 .
since the proportions of embryonic / fetal stem cells for the production of adipocytes may be altered 13 , it would be of interest to determine if the same relative proportional changes observed during in utero development are maintained throughout postnatal growth .
enhanced understanding here might point to means for augmenting specific populations of " desired " adipocyte - like cells to the exclusion of others 1 , 13 , 41 . postnatal development and growth .
the choice of a particular type of adipocyte - like cell to identify putative markers of cell physiology transitions from proliferative cells into cells that are conducting lipid metabolism needs to be carefully scrutinized and weighed 10 , 40 . indeed , sorting out the subpopulations of cells within adipose depots will allow more a discrete definition of such pathways , while allowing a determination of how " flexible " the population might be for other applications 17 , 41 , 47 .
inclusion of different sizes of mature adipocytes 48 , 49 , or their dedifferentiated progeny cells 47 in many of these types of studies will help define whether differentiation really exists , as traditionally defined , or whether we will need to reassess adipogenesis from all sides 12 , 29 , 40 .
for example adipocytes in avian adipose tissues do not exhibit de novo fatty acid synthesis while de novo fatty acid synthesis may occur at high rates in mammalian adipose depots 50 .
( human cells , 52 ) first suggested that adipose tissue contains a number of progenitor cells of several cell phenotypes .
most remarkably , these authors demonstrated that adipose tissue cells can give rise to adipocytes , osteoblasts , myoblasts , endothelial cells , and hematopoietic cells among other phenotypes 51 .
for example , is the cellularity of cells that form what appear to be lipid - filled adipocytes , as well as the mature adipocytes that might lose lipid and become proliferative - competent progeny cells , phenotypically " flexible ? " also , are isolated adipose - derived cells capable of forming many ( other ) types of cells ?
numerous studies have already provided evidence to address these questions , and adipocyte - derived cells from numerous sources appear capable of forming many other types of cells 5 , 6 , 10 - 12 , 17 , 26 , 30 , 33 , 41 , 48 , 53 - 59 . moreover , in general it has been estimated that adipose tissue may provide numerically higher amounts of stem cells 60 for use in a variety of applications than any other stem cell source of the body 26 , 47 , 61 , 62 .
also , the phenotype of adipose tissue - derived cell populations distinguishes adipose tissue depots from one another 51 .
collectively , cells derived from adipocyte populations may serve as a smorgasbord of progeny cells for biotechnology , tissue engineering , surgery , direct tissue repair ( regenerative medicine ) , and thus serve as a cell supermarket 10 , 12 , 26 , 37 , 46 , 53 , 59 - 61 , 63 - 67 .
the choice of species for the most promising model to study certain aspects of adipogenesis and trans / dedifferentiation by individual workers is likely highly dependent on the animal resources of an institution .
the availability of multiple species , in particular farm animals , would offer potentially a highly diverse / smorgasbord sources of cells . while such species ( in addition to rodent and cell lines ) are generally available at institutions with agricultural and veterinary colleges , such an approach may not be realistic for mainstream biomedical research centers . here the principal cell resources and infrastructure may only accommodate rodent tissues , human tissues and cell lines .
it would also appear that for commercial application , adipose tissue related cells from farm species would for the foreseeable future be available only as primary cells . in additions strategies and means
unfortunately unless there are overriding experimental model advantages for use of farm animal cells , the majority of workers in this field would continue to use rodent tissues , cell lines and available human adipose sources .
most likely if the majority of the work alluded to here would be done with rodents , then most of potential funding then would also be focused on these species .
this then will place the institutions with extensive animal resources , including farm animals at a strategic disadvantage to pursue this line of research from a comparative perspective .
the quantitative rate and extent ( success ) of cells derived from adipose depots to become transformed into other types of cells must be examined more closely .
not only is it important to determine if the cells can transdifferentiate , but whether there exist populations of these cells , whose proportions might be increased / decreased with lifestyle choices such as type of diet , amount of energy intake and level of exercise . in this respect , identification of the regulatory mechanism(s ) involved in transdifferentiation is essential for understanding the cell physiology involved 26.are some adipocyte subpopulations more capable of serving different and useful roles ?
for example , research data are presently not available to predict what type of transdifferentiated adipocyte - like cells ( and regulators ) would be most beneficial to aid bone repair in a clinical setting versus a different set of cells and regulators which would be more advantageous to applications such as cosmetic surgery.future studies dealing strictly with cells derived from adipose tissues to be used in clinical settings need to consider variables such as : a ) species and location of adipose depots from which such cells are to be derived , b ) a clear delineation of representatives of all populations of potential stem cells that may be isolated ( if possible ) from given adipose depots , c ) characterization of transdifferentiation potentials ( endogenous or artificially induced ) of harvested cells and d ) establish whether heterogeneous cells arising originally from adipose depots may be reprogrammed into lipid - assimilating adipocyte - like cellsconsiderable number of studies with human adipose tissue stromal / stem cell clearly demonstrated the nature and function of adipose tissue stromal / stem cell progenitors ( adsc ) of vascular entities like endothelial , perivascular and smooth muscle cells involved in angiogenesis and vasculogenesis .
a novel vascular stem cell ( vsc ) theory proposes that adsc are a mixed population of vsc with differential potential proportional to the angiogenic potential of the vasculature 68 .
the differential potential of vsc can range considerably in a continuous as opposed to a discrete fashion and can include vascular smooth cells , endothelial cells and adipocytes 68 .
adsc are likely vsc at various stages of differentiation toward becoming smooth muscle and endothelial cells . at earlier stages of differentiation vsc
can also differentiate in situ into host tissue - specific cell types such as adipocytes in their native tissue .
finally , depending on its differentiation potential at the time of isolation from the host tissue , an individual vsc may be induced to differentiate into additional cell types such as osteoblasts and chondrocytes .
therefore , adsc may exist in adipose tissue as a mixed population of vsc as opposed to strictly as adipocyte stem cells .
these emerging realities of cellular heterogeneity ( pluripotent vs. committed cells for example ) , differentiation and trans / de - differentiation potential need further investigation .
the quantitative rate and extent ( success ) of cells derived from adipose depots to become transformed into other types of cells must be examined more closely . not only is it important to determine if the cells can transdifferentiate , but whether there exist populations of these cells , whose proportions might be increased / decreased with lifestyle choices such as type of diet , amount of energy intake and level of exercise . in this respect , identification of the regulatory mechanism(s ) involved in transdifferentiation is essential for understanding the cell physiology involved 26 .
for example , research data are presently not available to predict what type of transdifferentiated adipocyte - like cells ( and regulators ) would be most beneficial to aid bone repair in a clinical setting versus a different set of cells and regulators which would be more advantageous to applications such as cosmetic surgery .
future studies dealing strictly with cells derived from adipose tissues to be used in clinical settings need to consider variables such as : a ) species and location of adipose depots from which such cells are to be derived , b ) a clear delineation of representatives of all populations of potential stem cells that may be isolated ( if possible ) from given adipose depots , c ) characterization of transdifferentiation potentials ( endogenous or artificially induced ) of harvested cells and d ) establish whether heterogeneous cells arising originally from adipose depots may be reprogrammed into lipid - assimilating adipocyte - like cells considerable number of studies with human adipose tissue stromal / stem cell clearly demonstrated the nature and function of adipose tissue stromal / stem cell progenitors ( adsc ) of vascular entities like endothelial , perivascular and smooth muscle cells involved in angiogenesis and vasculogenesis . a novel vascular stem cell ( vsc ) theory proposes that adsc are a mixed population of vsc with differential potential proportional to the angiogenic potential of the vasculature 68 .
the differential potential of vsc can range considerably in a continuous as opposed to a discrete fashion and can include vascular smooth cells , endothelial cells and adipocytes 68 .
adsc are likely vsc at various stages of differentiation toward becoming smooth muscle and endothelial cells . at earlier stages of differentiation vsc
can also differentiate in situ into host tissue - specific cell types such as adipocytes in their native tissue .
finally , depending on its differentiation potential at the time of isolation from the host tissue , an individual vsc may be induced to differentiate into additional cell types such as osteoblasts and chondrocytes .
therefore , adsc may exist in adipose tissue as a mixed population of vsc as opposed to strictly as adipocyte stem cells
. these emerging realities of cellular heterogeneity ( pluripotent vs. committed cells for example ) , differentiation and trans / de - differentiation potential need further investigation .
the general characteristics of stem cell development to committed and finally differentiated cells have been described .
presently we are focusing on the potential of adipose tissues as a source of stem and other recommitted cells for further study and future clinical applications .
unfortunately the extent of our understanding of stem cell biology is still too limited to envision how a rich source of such cells ( as adipose tissue ) may be utilized in therapeutic programs in future clinical settings . | adipose tissue is derived from numerous sources , and in recent years this tissue has been shown to provide numerous cells from what seemingly was a population of homogeneous adipocytes .
considering the types of cells that adipose tissue - derived cells may form , these cells may be useful in a variety of clinical and scientific applications .
the focus of this paper is to reflect on this area of research and to provide a list of potential ( future ) research areas . |
epidemiology can be defined as the study of the occurrence and distribution of disease and its determinants . in a broader approach ,
the areas of research in epidemiology include disease definition , occurrence , causation , outcome , management , and prevention .
the occurrence of a disease may be studied in relation to factors that can identify or predict the disease ( diagnostic factors ) or are thought to influence its occurrence ( eg , prognostic or etiologic factors ) .
furthermore , the association between a particular intervention and a change in the occurrence of the disease is of importance . in a recent review on rheumatic diseases , gabriel and michaud [ 3 ]
acknowledged that until recently , very few studies had been conducted on the epidemiology of gout . notwithstanding
the rising incidence [ 4 , 5 ] , the burden of disease associated with gout and the frequency of gout as a comorbidity in patients with multiple morbidities highlight the importance of gaining a better understanding of the etiology , pathology , and management of gout through epidemiologic studies .
this article focuses on the challenges of epidemiologic studies that aim to estimate the occurrence of gout in terms of prevalence or incidence .
such studies require large sample sizes , especially in heterogeneous populations . however , it is not only sample size that needs consideration when conducting epidemiologic studies on the occurrence of gout .
first , the definitions used in studies of gout are discussed , as well as the differences between diagnostic and classification criteria .
next , the criteria regularly used in epidemiologic studies are reviewed and critically appraised . finally , several additional challenges encountered when interpreting results of epidemiologic research on gout are addressed .
the gold standard to diagnose gout is to demonstrate the presence of monosodium urate monohydrate ( msu ) crystals in synovial fluid at the time the patient experiences a gout attack .
however , this is easier said than done in clinical practice , as gout patients are often seen by general practitioners or specialists other than rheumatologists , who rarely perform a synovial fluid analysis to demonstrate urate crystals .
reasons for nonperformance include lack of expertise , limited access to polarizing microscopes , lack of time , or the concern of getting a dry tap .
if a joint aspiration is performed , several difficulties remain , as both false - positive and false - negative results may occur . in some cases
, cholesterol crystals may appear as needle - shaped birefringent crystals [ 11 , 12 ] .
furthermore , it is debatable whether one can diagnose a patient as having gout when only one or two msu crystals are seen , or whether one should perform a second joint aspiration if no crystals are detected the first time . on the other hand , lumbreras et al
. showed that when observers are trained in crystal detection and identification , their results are usually consistent .
although synovial fluid examination remains best practice in clinical medicine , in the context of epidemiologic studies , this may not be feasible , especially in view of the intermittent nature of gout and the sample sizes that are needed in population studies . in studies ,
however , comparing their definitions , if they are provided at all , these terms seem to be used inconsistently by different authors . to facilitate the comparability of study results , clear definitions of the various manifestations and stages of gout therefore are needed .
[ 14 ] proposed key components of a standard definition of gout flares using the delphi methodology .
the final list of elements includes a swollen , tender , and warm joint ; patient self - report of pain and global assessment ; time to maximum pain level ; time to complete resolution of pain ; functional status ; and an acute - phase marker .
interestingly , although a core set of outcome domains to be used in clinical trials was proposed by omeract ( outcome measures in rheumatology clinical trials ) , the group did actually not provide a clear definition of chronic gout but agreed on serum urate , gout flare recurrence , tophus regression , joint damage imaging , health - related quality of life , musculoskeletal function , patient global assessment , participation , safety , and tolerability as core outcome domains to be assessed in trials on gout . for choi et al . , acute gout is typically intermittent , whereas chronic tophaceous gout develops after years of acute intermittent gout .
however , they point out that tophi can also be part of the initial presentation .
this is in line with the american college of rheumatology ( acr ) criteria for acute gout , which incorporate the item more than one attack of acute arthritis as well as suspicion of a tophus .
others state that after a certain undefined number of attacks , a patient has reached a stage called recurrent gout .
if a patient can not recover from the flares , it becomes chronic gout . in that case
some authors have a more inclusive definition of chronic gout , incorporating all patients who have had more than one attack of acute gout .
the underlying reasoning is that once a patient has had a flare , a persistent metabolic disorder exists .
in addition to these deliberations , one might think of gout as a continuum of increasing severity .
a first step would be to define the domains that are of importance when deciding on the severity of gout .
of interest is a recent study that explored which variables are associated with patients as opposed to physicians assessment of gout severity .
it was found that physicians base their judgment of severity on the presence of tophi , frequency of gout attacks during the past year , recent serum uric acid levels , and rheumatologist utilization , whereas patients judgment of severity is associated with concerns regarding gout during an attack and the time since the last gout attack . it will be a challenge to try to measure each of these domains and to define thresholds to distinguish levels of severity based on the selected domains .
this may also include addressing issues such as total load of uric acid and total load of tophi . in summary , the question remains whether intermittent or recurrent gout must be distinguished from chronic gout and , if so , at which point acute or recurrent gout develops into chronic gout .
can we speak of chronic gout after a number of attacks or after a specific period of recurrent attacks , or only when a patient has bone destruction and chronic synovitis , even during remission of acute flares ?
should we make a distinction between chronic gout and tophaceous gout , and should we distinguish different levels of severity of gout ?
currently , no clear answers to these questions exist , and the lack of insight into the natural course of gout among all patients complicates this issue .
some patients may not recall attacks of symptomatic gout , and patients with tophaceous gout may no longer experience acute attacks .
clearly , a need exists for consensus on definitions that help distinguish the different manifestations of the disease .
the title of this article may seem contradictory because in epidemiologic studies , no diagnostic criteria other than classification criteria are applied .
these criteria can be used to select patients for clinical ( interventional ) studies , to compare the results of clinical trials , or to assess the occurrence of a disease in epidemiologic studies .
in contrast to diagnostic criteria , classification criteria do not have the purpose of early detection of a disease in an individual patient . instead
, classification criteria are used to detect established cases . as for diagnostic tests , calculating the sensitivity and specificity assesses the usefulness of criteria .
sensitivity is the percentage of individuals with a certain disease correctly classified as ill ( true positives ) .
the percentage of individuals without the disease correctly labeled as not ill is the specificity ( 100% - percentage of false positives ) . if the sensitivity and specificity of criteria are both 100% , diagnostic and classification criteria are the same .
note that diagnostic criteria would require sufficient sensitivity in early stages of the disease to enable early diagnosis .
however , the nature of medicine makes it unlikely that there will ever be tests that offer 100% sensitivity and specificity .
therefore , misclassification poses a challenge , and the type of misclassification that is least desirable will depend on the setting in which the test is applied . in health care
, physicians must identify which disease a patient has rather than whether a disease exists at all .
they do not want to misdiagnose a patient and may prefer high sensitivity against acceptable specificity .
in contrast , in epidemiologic studies in large populations , to study homogeneous groups that are likely to have the diagnosis of interest but do not include many false positives , the researchers must balance specificity and sensitivity and will often sacrifice part of sensitivity against better specificity .
of course , any misclassification , which is common in classification criteria , is undesirable .
an approach to minimize misclassification is the use of cut - off points . in deciding on a cut - off point ,
one has to choose between a sensitive approach involving false positives and a more specific approach that results in a more homogeneous group and more false negatives .
classification criteria often are developed by comparing groups with the disease of interest with control patients having other ( usually related or resembling ) diseases that should be taken into account in the differential diagnosis .
however , one must keep in mind that if these criteria are applied in population studies , the positive predictive value ( ppv ) may decrease , especially when the prevalence of the disease of interest is low .
the ppv is defined as the number of individuals with a true positive test result divided by all individuals with a positive test result ( true positives + false positives ) . in other words
, it indicates the probability that in case of a positive test , the patient truly has the specified disease .
the value of ppv depends on the prevalence of the disease of interest in the particular setting and will decrease when the prevalence goes down , due to the increasing number of false positives .
thus , when applying the criteria for gout , a disease with a relatively low prevalence at the general population level , it is important to keep in mind that the estimated prevalence may be overestimated due to the unintended inclusion of false positives .
in this section , criteria to assess the prevalence of gout in epidemiologic studies are described . for this purpose ,
the eular ( european league against rheumatism ) criteria , which are purely diagnostic criteria and intended for use in individual patients with arthritis and not for use in groups , are excluded . in 1963 , the rome criteria for gout were proposed during a symposium on population studies ( table 1 ) . these and the 1966 new york criteria , which are a modification of the rome criteria , are based on expert opinion and aimed for application in epidemiologic studies ( table 1 ) .
they rely heavily on the presence of tophi and the observation of msu crystals in synovial fluid , which causes some feasibility issues .
the rome criteria have only been used in two population studies assessing the prevalence or incidence of gout , once as interview and once as questionnaire , and both times in combination with a physical examination .
the new york criteria have been used in several population studies in the same way as the rome criteria [ 2124 ] .
table 1classification criteria for goutrome criterianew york criteriaamerican college of rheumatology criteriatwo of the following 4 criteria must be present to make a diagnosis of gout : urate crystals in synovial fluid or tissue or presence of at least 2 of the following : preliminary criteria for the classification of the acute arthritis of primary gout1 .
serum uric acid level 7.0 mg / dl in men , or 6.0 mg / dl in women1 . history or observation
of at least 2 attacks of painful limb swelling with remission within 12 weeka .
history of attacks of painful joint swelling of abrupt onset with remission within 12 week4 .
history or observation of a good response to colchicines ( major reduction in objective signs of inflammation within 24 h of onset of therapy)1 . more than 1 attack of acute arthritis2 .
joint fluid culture negative for organisms during attacks classification criteria for gout it should be noted that for use in population surveys , the items that make up criteria likely need to be rephrased into questions that are answerable by patients using questionnaires or participating in interviews .
currently , the most frequently used methods to identify people with gout in epidemiologic studies are the acr criteria
the acr criteria for gout have been developed to achieve a uniform system for reporting and comparing data from studies ( table 1 ) .
they have been developed by comparing different sets of criteria among gout patients and patients with classic rheumatoid arthritis of 2 years or less duration , definite or classic rheumatoid arthritis of more than 2 years duration , pseudogout , or acute septic arthritis .
all have been diagnosed by rheumatologists . as such , the acr criteria for gout focus on acute arthritis of primary gout and can be used in single patients as well as in population surveys . in large studies on the occurrence of gout ,
the acr criteria are often applied by interviewing patients with or without a standardized questionnaire , or by chart review of medical records .
although the acr criteria were developed for the diagnosis of acute gout , they also have been used to identify so - called chronic gout , when patients fulfill the item tophi or radiographic abnormalities .
compared with the rome and new york criteria , the acr criteria rely less on the presence of tophi or identification of msu crystals and even allow classification based on clinical criteria alone .
applied the acr , new york , and rome criteria in patients who had joint effusions in the setting of a rheumatology clinic .
they asked patients whether they had experienced any of the clinical features of these three sets of criteria .
the researchers found the highest specificity ( 89% ) and ppv ( 77% ) for the rome criteria .
the acr criteria ( 6 of 12 clinical items ) had 70% sensitivity and 79% specificity and a ppv of only 66% .
clearly , one should not extrapolate such findings to an epidemiologic population study , because the ppv varies with the pretest probability , which , as mentioned previously , is highly dependent on the prevalence of the disease .
compared the acr criteria with synovial fluid analysis as a gold standard in monoarthritic patients presenting to primary care .
they found a ppv and a sensitivity of 80% , while specificity was 64% . according to janssens et al .
, these findings stress the importance of interpreting with caution the results of gout studies that made use of the acr criteria . a common method used to estimate the prevalence of gout is the use of large medical databases that have registered diseases by icd-9 coding .
examples of databases used in gout research include medical patients record systems , administrative claims , and insurance programs .
advantages of such databases are the large numbers available at low expenses and the efficient time investment .
a disadvantage is that it remains unclear how the diagnosis was made by the variety of health professionals and how to generalize the results because the denominator is often unclear .
evaluated the possibility of documenting the accuracy of the icd-9 code for gout in three databases ( national patient care database , pharmacy benefits management database , and the clinical and administrative database ) by identifying patients with two icd-9coded encounters for gout during a 6-year time period .
they found that identifying the items of the acr , new york , or rome criteria in medical records could not validate the majority of gout diagnoses recorded by icd-9 .
this discrepancy may be caused by inadequate documentation in medical records , inaccurate diagnostic coding , or the inappropriateness of current criteria . according to malik
harrold et al . analyzed a random sample of medical records of patients with two or more coded diagnoses of gout from four managed care plans .
the ppv of two or more ambulatory claims ( during a time period of 5 years ) for a diagnosis of gout was assessed using the investigators rating of the presence or absence of definite or probable gout as the gold standard .
substantial improvement in the ppv was not achieved by increasing the number of visits to three or four .
explanations for the disappointing ppv include the ambiguity of a diagnosis of gout compared with , for example , a more firm diagnosis of myocardial infarction ; the assignation of an icd code before the diagnosis was firmly established ; the underutilization of synovial fluid analysis ; and inadequate documentation in medical records .
self - reported disease , sometimes completed with information from other medical sources , is often applied in epidemiologic studies of gout .
however , it is difficult to distinguish between questionnaires that inquire about physician - diagnosed gout and questionnaires that inquire about manifestations typical of gout based on the existing criteria described above . furthermore , such questionnaires vary in the time frame in which gout occurred , which can be one or more attacks at some point in the past or several attacks in a specific ( limited ) period of time preceding the survey .
miller et al . analyzed the agreement between self - reported diseases and icd-9 coding .
however , only 50% of self - reported cases of arthritis could be confirmed by icd coding .
reasons for this lack of agreement may be that responders have interpreted a question incorrectly or have recalled a diagnosis that was not actually established or was recalled inaccurately .
however , if patients are not receiving medication or other treatment , the diagnostic code for a certain condition may not be written down in the record .
miller et al . pointed out that acute events that occurred in the past and conditions that are episodic in nature are not always captured if the reviewing period is too short .
although self - completed questionnaires may cover a large population in a relatively short time period at low cost , the downside is a possible low response rate .
using an interview approach , it is possible to ensure that all questions are answered in the correct manner .
reported that the agreement between a face - to - face interview and a self - administered questionnaire was moderate ( , 0.61 ) .
less serious , less defined , or less persistent diseases such as gout may be perceived by patients as not being important enough to report in questionnaires . of interest
might be the diagnostic rule for acute gouty arthritis recently developed by janssens et al .
based on validated clinical variables using synovial fluid analysis as a reference test , a multivariate logistic regression model was developed .
hereafter , they developed two models based on external knowledge and availability of the tool in clinical practice .
their final model includes seven variables : male sex , previous patient - reported arthritis attack , onset within 1 day , joint redness , first metatarsophalangeal joint involvement , hypertension , or one or more cardiovascular diseases and serum uric acid level exceeding 5.88 mg / dl ( 0.35 mmol / l ) .
although developed for use in primary care , the diagnostic rule may be useful in a research setting .
however , it is not known how well this model performs in a population study ( work in progress ) .
in addition to the above considerations that are critical in the appraisal of data on the occurrence of disease , several other issues merit consideration when appraising the results of such studies . first is the question of which type of epidemiologic measure of occurrence was applied .
the nature of disease will influence the relevant study design and measure of occurrence that is most informative . as acute flares of arthritis
mainly characterize gout , the point prevalence estimate ( table 2 ) is not likely of primary interest .
in fact , in a cross - sectional population study , the chance that someone will suffer from a gout attack at exactly the time of the survey is low . in this case ,
the period prevalence , which represents individuals who experienced one or more episodes over a specified period preceding the survey ( table 2 ) , will be more informative . only if chronic gout would be described in terms of persistent joint inflammation , presence of irreversible joint damage , or presence of tophi would the point prevalence be interesting .
table 2conceptual framework of incidence and prevalence in studies of goutincidence cumulative incidenceto be assessed in a cohort : number of new cases of gout per year divided by the population at risk ( ie , all cohort members who at the initiation of the cohort or at the start of the year of incidence assessment had never experienced any manifestation of gout ) incidence densityto be assessed in a dynamic population ( eg , the inhabitants of new york city ) : number of new cases of gout per year divided by the number of person - years of individuals at risk .
one person - year is defined as 1 person who is at risk for a 1-year period ( eg , if an individual gets gout after 3 months , he or she is counted as 0.25-person years in the denominator ) .
thus , the denominator of incidence density ( ie , the number of person - years in a dynamic population ) is not only determined by the changing size of the total population ( eg , accounting for individuals entering and leaving the municipality of new york , as well as newborns and deaths ) but also by the number of hitherto - healthy individuals who for the first time get gout during the observation period and are therefore ( from that moment onward ) no longer at risk of newly getting goutprevalence point prevalencenumber of cases of gout in the study population at a given point in time divided by the total study population .
this comprises the ( usually few ) individuals who suffer from an acute attack of gout at the concerned point in time together with all those who then have chronic ( tophaceous or nontophaceous ) gout period prevalencenumber of cases of gout in the study population during a specified period of time divided by the mean size of the total study population over the concerned period .
this comprises all individuals who have experienced an acute attack of gout during that period together with all cases of chronic ( tophaceous or nontophaceous ) gout conceptual framework of incidence and prevalence in studies of gout another important concept in epidemiologic studies is the incidence or incidence rate , which refers to the number of new cases of gout in a population ( table 2 ) .
cumulative incidence refers to new cases of gout per year divided by all members of a cohort ( ie , a closed population ) who are at risk ( ie , who never experienced any signs of gout before the observation period ) .
in contrast , incidence density refers to new cases of gout per person - year in a dynamic population , such as the inhabitants of a region or municipality ( table 2 ) .
it is also important to carefully consider the population that has been studied . as for any epidemiologic study
, the sample should be a correct representation of the population of interest ; this requires insight into the sampling frame and participation rate
. the participation rate should be at least as high as 80% ; however , a rate between 60% and 80% with a description of the nonresponders is often considered acceptable .
furthermore , to guarantee the representativeness of the samples in studies on gout , it is important to take into account sources of ( selection ) bias , such as the age , sex , and race of the study population . determining the prevalence in a preponderant older male population will overestimate the occurrence of gout in the general population .
one also should be aware of confounding factors such as alcohol consumption , body mass index , and comorbidities .
obesity , diabetes , and hypertension are common among patients with gout , and the prevalence of the metabolic syndrome is higher than in patients without gout [ 34 , 35 ] .
although the pathophysiology of gout is relatively well - understood , it is surprisingly difficult to define good classification criteria for use in large population studies to validly assess the prevalence and burden of gout
. this is partially due to the nature of the disease , which is typically intermittent , which limits the ability to use msu crystals as the gold standard in large epidemiologic studies . also challenging
is the absence of clear insight into the natural course of the disease , which would require better definitions of the manifestations and stages of gout .
although there is general agreement that gout is likely the result of a longstanding metabolic disorder that eventually leads to clinically manifest gout , it is less clear how many patients will progress to tophaceous gout and develop joint damage . in view of the aforementioned considerations , literature data on the prevalence of gout
are surprisingly consistent . in developed countries , estimates vary between 1% and 2% . nevertheless , as discussed previously , different levels of misclassification must have occurred in these studies .
this will hamper interpretation of the results , especially in light of risk factors and comorbidities associated with gout .
more precise estimates of the prevalence and burden of gout require addressing the validity of classification criteria and proper definitions of the various manifestations and stages of severity of gout .
mcadams et al . reported recently that self - report of physician - diagnosed gout has good reliability and sensitivity and that this method may seem appropriate for epidemiologic studies . | large epidemiologic studies of gout can improve insight into the etiology , pathology , impact , and management of the disease .
identification of monosodium urate monohydrate crystals is considered the gold standard for diagnosis , but its application is often not possible in large studies .
therefore , under such circumstances , several proxy approaches are used to classify patients as having gout , including icd coding in several types of databases or questionnaires that are usually based on the existing classification criteria .
however , agreement among these methods is disappointing .
moreover , studies use the terms acute , recurrent , and chronic gout in different ways and without clear definitions .
better definitions of the different manifestations and stages of gout may provide better insight into the natural course and burden of disease and can be the basis for valid approaches to correctly classifying patients within large epidemiologic studies . |
a 66-year - old woman with sjgren syndrome presented with a 5-year history of bilateral shoulder pain that was significantly worse on the right than the left side .
the pain was reported to be constant in nature but worse at night , and completely prohibited activities above shoulder height .
examination of the right shoulder revealed a globally restricted range of movement at the glenohumeral joint .
treatment with copeland resurfacing hemiarthroplasty was performed in june 2007 through an anterosuperior ( mackenzie ) approach .
. 1 interrupted pds ( polydioxanone ) sutures were used for reattachment of the anterior border of the osteo - periosteal sleeve to the acromion .
the sutures were placed transosseously through the acromium on one side and through the deltoid fascia , incorporating an osteo - periosteal sleeve , on the other [ figure 2 ] .
preoperative radiograph demonstrating severe osteoarthritis radiograph taken in the immediate postoperative period showing a copeland resurfacing hemiarthroplasty passive elevation past 90 could be achieved on the first postoperative day .
a shoulder immobilizer was worn for 3 weeks and the patient was instructed to avoid active elevation .
the patient had returned to aerobics and table tennis by the 4 postoperative month , at which point increased pain in the shoulder was reported . at the end of the 1 postoperative year , significant impingement symptoms were reported and x - rays demonstrated a new bony spur on the anteroinferior edge of the acromion [ figure 3 ] .
an ultrasound scan showed a bony spur indenting the supraspinatus , but there was no accompanying cuff tear .
the pain continued to worsen and diagnostic arthroscopy was performed in july 2009 , which confirmed the presence of the spur at the anteroinferior edge of the acromion and excluded the presence of significant glenoid arthrosis or adhesions as contributing factors to the patient 's symptoms .
radiograph at 1 year showing new bony spur on the inferior surface of the acromion appearance following arthroscopic resection of the spur
the mackenzie anterosuperior approach has been popularized for several types of shoulder arthroplasties . in this approach the anterior deltoid is commonly reflected with an osteo - priosteal sleeve .
we postulate that the bony spur responsible for the impingement symptoms in our patient was formed by malunion of the osteo - periosteal sleeve . at the time of arthroscopy ,
a sharp spur of bone of the same size and shape as the osteo - periosteal sleeve was found to be united in the correct position on the anterior acromion but , from its shape , it had apparently rotated 90 from the original orientation .
the onset of symptomatic impingement along with new x - ray findings at 4 months postoperatively is consistent with rotation of the fragment during healing following the patient 's return to sporting activity .
full resolution of the patient 's symptoms following resection of the spur also supports this conclusion .
intra - articular factors are the most common reasons for postoperative pain following copeland resurfacing hemiarthroplasty and are due to the nonanatomical shape of the prosthesis , which does not accurately recreate the original shape of the humeral head .
as our patient was initially pain free with good shoulder function , we carried out other investigations , including diagnostic arthroscopy , to look for other causes for her pain preoperative investigation to establish the diagnosis of subacromial impingement could include an injection of local anesthetic under aseptic technique into the subacromial space .
immediate pain relief would be convincing evidence of the existence of subacromial impingement . to the best of our knowledge ,
this is the first report of formation of an acromial spur following the mackenzie approach .
complications of the copeland resurfacing hemiarthroplasty have been reported as infection , aseptic loosening , periprosthetic humeral fracture , and osteolysis .
subacromial impingement has been reported in patients after this procedure and the symptoms have necessitated subacromial decompression , but this has not previously been reported to be associated with a malunited osteo - periosteal sleeve . nonunion ( or fibrous union ) has been reported by previous authors following surgical access using the mackenzie approach but they did not report symptomatic subacromial impingement . in our patient , simple arthroscopic excision of the bony spur proved curative .
this case has altered our practice , as we have now abandoned the osteoperiosteal sleeve technique and instead opt for subperiosteal dissection of the anterior deltoid .
we conclude that the cause for the delayed , severe impingement symptoms in this patient following successful copeland resurfacing hemiarthroplasty was malunion of the osteo - periosteal sleeve that was made as part of the mackenzie approach . all symptoms resolved following arthroscopic resection of the spur . | we report the case of a patient with end - stage osteoarthritis who received a successful copeland resurfacing hemiarthroplasty through a mackenzie anterosuperior approach , which involves taking the anterior portion of the deltoid attachment from the acromion along with an osteo - periosteal sleeve .
the patient went on to develop severe subacromial impingement symptoms 4 months postoperatively .
x - rays revealed a large anteroinferior acromial osteophyte that had not been present preoperatively and was deemed to represent a malunited osteo - periosteal sleeve from the mackenzie approach . |
a 65-year - old woman was admitted to our clinic with complaints of sudden , painless , decrease in vision , and sectoral visual field defect in the left eye for the past 3 days . on examination ,
fundus examination of the left eye showed a patchy area of partially faded retinal whitening in the inferotemporal quadrant of the macula , just encroaching into the fovea .
fluorescein angiogram of the left eye showed a delay in arterial filling in the affected vessels [ fig .
she had no history of amaurosis fugax , diabetes mellitus , coronary artery disease , or stroke .
a fundus examination of the right eye showed an emboli and segmentation in the inferotemporal artery . due to these findings , cardiology consultation , carotid doppler ultrasound , echocardiography and
four days after the initial examination , she was presented to our clinic again with a history of sudden loss of vision in her right eye for the last 4 h. visual acuity in the right eye was reduced to perception of light only . the right eye fundus examination demonstrated a diffuse disc pallor , severe attenuation of retinal arteries , retinal edema , and a cherry - red spot appearance on the macula [ fig .
the red - free photograph greatly accentuates the retinal whitening surrounding the macula and with segmentation of blood columns in inferotemporal retinal artery because of the multiple emboli plaques [ fig .
she was immediately given a vigorous digital ocular massage , breathing carbogen ( 95% oxygen , 5% carbon dioxide ) , started on intra - venous mannitol and systemic carbonic anhydrase inhibitor .
unfortunately , visual acuity in the right eye was not changed after the treatment . in the cardiovascular evaluation , there was a fibro - fatty plaque in the right internal carotid artery at carotid doppler ultrasonography .
serum cholesterol level was 231 mg / dl and serum low - density lipoprotein level was 166 mg / dl .
an urgent erythrocyte sedimentation rate , c - reactive protein , and complete blood count were normal .
however , there was a neovascularization of optic disc in the right eye . optical coherence tomography ( oct ) revealed diffuse thickening of the inner retinal layers .
increased reflectivity was noted in the inner retinal layers from the surface of the retina to the photoreceptor layers .
3 . ( a ) the right eye fundus photo reveals whitening of the inferior retina with a calcified plaque at the disc margin within the inferotemporal artery . ( b - d ) the left eye fluorescein angiogram shows delay in arterial filling in the affected blood vessels and late leakage from the embolus site ( a ) the right eye fundus photo shows a disc pallor , attenuation of retinal arteries , retinal edema , and a cherry - red spot appearance of the macula .
( b ) the right eye red - free photograph shows segmentation of the blood columns because of the multiple emboli plaques ( a ) fundus photograph in the right eye in the first month .
retinal arterial occlusions are divided into central and branch groups depending on the precise site of occlusion .
generally , emboli may originate from atheromatous plaques in the ascending aorta or carotid arteries , as well as calcified heart valves .
emboli to the retinal circulation may originate at any point in the proximal circulation from the heart to the ophthalmic artery .
emboli were also visible in our patient in the left eye and it probably originated from the plaque in the calcified mitral and aortic valves . in crao , potential embolic sources are found in less than 40% of the cases . however , most common etiologic association is the significant ipsilateral carotid artery disease which is present in approximately one - third of all the patients . in our patient ,
the emboli in the right eye probably originated from the atheromatous plaque in the ipsilateral internal carotid artery .
the existence of emboli must be considered carefully during the retinal examination for the patients with temporary or permanent visual impairments .
if emboli are present , this should be urgently evaluated for cardiovascular disorder by a cardiologist . retinal artery occlusion
is also associated with hyper - tension , diabetes , vascular spasm as in migraines , hyper - coagulable states , glaucoma , or trauma .
multiple studies have shown that increased morbidity and mortality in the patients with retinal arterial emboli . as there is a strong association with fatal systemic disease and risk of blindness
, all patients who suffer from retinal artery occlusion should undergo a detailed and rapid systemic evaluation . because the accompanying visual loss tends to be severe and permanent , diagnosis and systemic treatment of these patients are critical . eliminating the risk factors is important for well - being of the unaffected eye .
accordingly , aspirin was prescribed 80 mg a day for reducing the risk of strokes , heart attacks , and the risk of blood clots in the body .
in addition , 20 mg a day of rosuvastatin was added to her medications for the diagnosis of hypercholesterolemia .
rosuvastatin is a member of the drug class of statins , they used to treat high cholesterol and prevent cardiovascular disease .
also , diet , exercise , and weight reduction were recommended as a part of the treatment .
furthermore , 80 mg of valsartan and 12.5 mg of hydrochlorothiazide fixed - dose combinations were prescribed once daily for the hypertension by the cardiology team .
there has been recent interest shown in the use of thrombolytic therapy , delivered either intravenously or intra - arterially by direct catheterization of the ophthalmic artery .
two large reviews have recommended that thrombolysis in the treatment of crao may recover the visual acuity with few serious complications .
however , data from a large multicenter randomized studies have failed to point out an improvement in the visual acuity .
this treatment must be performed within a short time period , probably within 6 h of symptom onset . due to the lack of technical infrastructure in our hospital , we were not able to use this treatment .
as a conclusion , it is an unfortunate condition to have crao on the right eye while investigating the reasons and risk factors for brao on the left eye in this patient .
detailed and rapid systemic evaluation and cardiologic preventions were altered because of the risk of death and complete occlusion on her left eye .
the patient was reviewed 2 years later which has proved the same including her vision and her systemic condition has been stable . | a 65-year - old woman was admitted to our clinic with complaints of sudden , painless , decrease in vision , and sectoral visual field defect in the left eye and later presented to our clinic again with a history of sudden loss of vision in her right eye . in this case
study we reported that the patient had branch retinal artery occlusion ( brao ) in the left eye and at the same time progressing central retinal artery occlusion ( crao ) in the right eye . |
s. aureus , particularly methicillin resistant isolate ( mrsa ) , is an important cause of hospital and community acquired infection throughout the world .
the resistance to methicillin belongs to a penicillin - binding protein encoded by a mobile genetic element called the methicillin - resistant gene ( meca ) ( 1 ) . increasing its resistance to antibiotics is limited to those available antibiotics which are prescribed for treatment of different infections caused by this bacterium ( 2 ) .
macrolide , lincosamide and streptogramin type b ( mlsb ) antibiotics such as erythromycin , clindamycin and streptogramin b are usually used in the treatment of infections , particularly skin and soft tissue infections .
clindamycin is a noteworthy choice in the treatment strategies for various reasons ; 1 ) it has high tissue penetration ( except for the central nervous system ) , 2 ) good oral absorption makes it appropriate for outpatient therapy , 3 ) clindamycin can be used as a choice antibiotic in patients suffer from allergy to penicillin .
however , resistance to these antibiotics is significantly increasing all over the world ( 3 , 4 ) .
one mechanism can occur through methylation of their ribosomal site which is mediated by the presence of erythromycin resistance methylase ( erm ) genes .
methylases reduce binding of mlsb antibiotics to the target site in the 50s ribosomal subunit .
erma and ermc are two common genes responsible for the mlsb resistance in s. aureus .
most of the bacteria targeted by macrolides and lincosamides , including gram - positive species , spirochetes , and anaerobes , express erm methylases .
erm(a ) genes are frequently spread in methicillin resistant isolates and are generated by tn554 transposons while erm(c ) genes are mostly responsible for erythromycin resistance in methicillin susceptible isolates and are carried by plasmids
. the second mechanism can occur by drug efflux typically mediated by the atp binding cassette ( abc ) transporter msra and the other mechanism is drug modification by two enzymes that confer resistance to macrolides and lincosamides such mph(c ) or lnu(a ) , respectively ( 57 ) . despite the high incidence rate of mlsb resistant staphylococci , especially among mrsa ( 810 ) , currently there is a little information available on the incidence and types of these resistant bacteria in iran ( 11 ) . the present study aimed to provide information regarding the prevalence of mlsb resistant s. aureus isolates in tehran , iran .
in this descriptive study , a total of 101 clinical isolates of s. aureus were collected from hospitalized patients in five hospitals in tehran , iran ( december 2012 to april 2013 ) .
isolates were obtained from different clinical specimens including wounds , respiratory tract , urine , blood , sterile body fluids and abscesses .
isolates were characterized as s. aureus by standard microbiological methods including gram staining , catalase test , slide and tube coagulase test , growth on mannitol salt agar and deoxy ribonuclease test ( 12 ) .
all detected s. aureus isolates were stored in nutrient broth plus 20% glycerol at 70 c until study time .
determination of mlsb phenotypes was performed by the use of d - test as described previously ( 13 ) and according to the guidelines of clinical and laboratory standards institute ( clsi ) ( 14 ) .
three antibiotic resistance phenotypes were determined ; isolates containing constitutive mlsb ( cmlsb ) which were resistant to both erythromycin and clindamycin , isolates containing inducible mlsb ( imlsb ) which were resistant to erythromycin but sensitive to clindamycin and isolates containing the ms phenotype which showed resistance to merely macrolide and streptogramin b. a special disk diffusion procedure , d - test , was developed for discrimination of imlsbs . in imlsb
resistant isolates , resistance to clindamycin was induced by diffusion of erythromycin through the agar which led to flattening of the clindamycin zone of inhibition closest to the erythromycin disk ( a d - shaped zone ) while ms phenotype contained isolates forming a circular zone around the clindamycin disk .
induction test in cmlsbs included two phenotypes ; resistant ( r ) and hazy d zone ( hd ) . in r phenotype
in contrast , in hd phenotype , in addition to the observed bacterial growth at the presence of erythromycin , two zones of growth could be observed around the clindamycin disk .
outer zone had a light and hazy growth extending to the clindamycin disk and a more dense bacterial growth in the inner zone which was blunted proximal to the erythromycin disk as observed in phenotype d. erythromycin resistant isolates with flattening of the inhibition zone around the clindamycin disk were considered as positive for imlsb .
induction test description in imlsb included two phenotypes ; d and d. in d phenotype a d shaped clear zone was seen around the clindamycin disk proximal to the erythromycin disk while in d phenotype , in addition to the observed d shaped zone around the clindamycin disk , small colonies were grown at the inhibition zone of the clindamycin disk .
bacterial growth at the presence of erythromycin and circular clear zone around clindamycin disk was considered as ms positive and negative phenotype in induction test ( 13 ) .
detection of mrsa was performed by cefoxitine disk ( 30 g ) on the mueller hinton agar ( merck , england ) plate according to the clsi guidelines ( 14 ) .
all antibiotic disks were purchased from mast co , uk and s. aureus atcc 25923 was used as a standard strain .
bacterial genomic dna was extracted according to the method described previously ( 15 ) . in brief , five colonies from overnight incubated brain heart infusion agar plates ( merck , england ) were suspended in 300 l sterile distilled water and were heated at 100 c for 15 min .
after centrifugation at 14,000 rpm ( 10 min ) , the supernatant was used as the template dna in pcr .
multiplex pcr was performed for detection of erma and ermc genes whereas meca and msra genes were detected by single pcrs ( 1517 ) .
sequence of each primer and the reference for each pcr has been shown in table 1 .
amplification was performed in a final volume of 25 l containing 0.5 l of each primer ( 25 pmol ) , 1x pcr buffer , 1.5 mm mgcl2 , 0.2 mm dntp mix , 5 l of template dna and 1.5 u of taq dna polymerase .
pcr products were electrophoresed on 1% agarose gel ( roche , germany ) at 100 volts and later stained with ethidium bromide solution to see the amplified dna fragments under gel documentation system ( uv tech , uk ) with a molecular size marker ( 100 bp ladders , fermentas , lithuania ) .
resistant s. aureus isolates ( erma , ermc , and msra gene positive strains , courtesy of dr .
data were summarized using mean standard deviation ( sd ) and assurance intervals for the microbiological and demographic characteristics .
all analyses were performed using spss ( v. 20 , chicago , usa ) using fisher exact or chi - square tests and p values 0.05 were considered statistically significant .
in this descriptive study , a total of 101 clinical isolates of s. aureus were collected from hospitalized patients in five hospitals in tehran , iran ( december 2012 to april 2013 ) .
isolates were obtained from different clinical specimens including wounds , respiratory tract , urine , blood , sterile body fluids and abscesses .
isolates were characterized as s. aureus by standard microbiological methods including gram staining , catalase test , slide and tube coagulase test , growth on mannitol salt agar and deoxy ribonuclease test ( 12 ) .
all detected s. aureus isolates were stored in nutrient broth plus 20% glycerol at 70 c until study time .
determination of mlsb phenotypes was performed by the use of d - test as described previously ( 13 ) and according to the guidelines of clinical and laboratory standards institute ( clsi ) ( 14 ) .
three antibiotic resistance phenotypes were determined ; isolates containing constitutive mlsb ( cmlsb ) which were resistant to both erythromycin and clindamycin , isolates containing inducible mlsb ( imlsb ) which were resistant to erythromycin but sensitive to clindamycin and isolates containing the ms phenotype which showed resistance to merely macrolide and streptogramin b. a special disk diffusion procedure , d - test , was developed for discrimination of imlsbs . in imlsb
resistant isolates , resistance to clindamycin was induced by diffusion of erythromycin through the agar which led to flattening of the clindamycin zone of inhibition closest to the erythromycin disk ( a d - shaped zone ) while ms phenotype contained isolates forming a circular zone around the clindamycin disk .
induction test in cmlsbs included two phenotypes ; resistant ( r ) and hazy d zone ( hd ) . in r phenotype
in contrast , in hd phenotype , in addition to the observed bacterial growth at the presence of erythromycin , two zones of growth could be observed around the clindamycin disk .
outer zone had a light and hazy growth extending to the clindamycin disk and a more dense bacterial growth in the inner zone which was blunted proximal to the erythromycin disk as observed in phenotype d. erythromycin resistant isolates with flattening of the inhibition zone around the clindamycin disk were considered as positive for imlsb .
induction test description in imlsb included two phenotypes ; d and d. in d phenotype a d shaped clear zone was seen around the clindamycin disk proximal to the erythromycin disk while in d phenotype , in addition to the observed d shaped zone around the clindamycin disk , small colonies were grown at the inhibition zone of the clindamycin disk .
bacterial growth at the presence of erythromycin and circular clear zone around clindamycin disk was considered as ms positive and negative phenotype in induction test ( 13 ) .
detection of mrsa was performed by cefoxitine disk ( 30 g ) on the mueller hinton agar ( merck , england ) plate according to the clsi guidelines ( 14 ) .
all antibiotic disks were purchased from mast co , uk and s. aureus atcc 25923 was used as a standard strain .
bacterial genomic dna was extracted according to the method described previously ( 15 ) . in brief , five colonies from overnight incubated brain heart infusion agar plates ( merck , england ) were suspended in 300 l sterile distilled water and were heated at 100 c for 15 min .
after centrifugation at 14,000 rpm ( 10 min ) , the supernatant was used as the template dna in pcr .
multiplex pcr was performed for detection of erma and ermc genes whereas meca and msra genes were detected by single pcrs ( 1517 ) .
sequence of each primer and the reference for each pcr has been shown in table 1 .
amplification was performed in a final volume of 25 l containing 0.5 l of each primer ( 25 pmol ) , 1x pcr buffer , 1.5 mm mgcl2 , 0.2 mm dntp mix , 5 l of template dna and 1.5 u of taq dna polymerase .
pcr products were electrophoresed on 1% agarose gel ( roche , germany ) at 100 volts and later stained with ethidium bromide solution to see the amplified dna fragments under gel documentation system ( uv tech , uk ) with a molecular size marker ( 100 bp ladders , fermentas , lithuania ) .
resistant s. aureus isolates ( erma , ermc , and msra gene positive strains , courtesy of dr .
data were summarized using mean standard deviation ( sd ) and assurance intervals for the microbiological and demographic characteristics .
all analyses were performed using spss ( v. 20 , chicago , usa ) using fisher exact or chi - square tests and p values 0.05 were considered statistically significant .
s. aureus isolates were isolated from wound ( n=29 ) , urine ( n=25 ) , respiratory tract ( n=14 ) , sterile body fluids ( n=16 ) , blood ( n=11 ) and abscesses ( n=6 ) .
these isolates were obtained from the patients admitted to internal medicine ( 48.0% ) , intensive care units ( 28.0% ) , infectious diseases ( 16.5% ) and surgery ( 7.5% ) .
demographic characteristics showed that there were 64 males and 37 females with the mean age of 52.57 ( 21.15 ) years .
the distribution of mlsb resistance phenotypes based on the studied genes is illustrated in table 2 . among 101
s. aureus isolates , 58 ( 57.4% ) were resistant to methicillin and 57 ( 56.4% ) were reported as erythromycin resistant .
forty nine ( 84.4% ) of mrsa isolates and 8 ( 18.6% ) methicillin susceptible s. aureus ( mssa ) isolates were resistant to erythromycin , respectively .
the prevalence of cmlsb , imlsb and ms resistance phenotypes among erythromycin resistant isolates were 71.9 , 26.3 and 1.7% , respectively .
meca gene was detected in all mrsa isolates . among 41 isolates with cmlsb resistance phenotype ,
38 isolates were mrsa and 10 out of 15 isolates with imlsb resistant phenotypes were mrsa .
thirty three isolates in the cmlsb group were positive for both erma and ermc genes aside from the presence or absence of msra gene .
eight isolates of imlsb category contained one of erma or ermc genes and none of them had msra gene .
ms phenotype was only found in one mrsa isolate and this isolate did not harbor erm genes or msra gene . the most common genes in erythromycin resistant isolates were erma ( 66.6% ) , ermc ( 63.1% ) and msra ( 3.5% ) , respectively .
none of the 8 mssa erythromycin resistant isolates had erma , ermc or msra gene .
distribution of resistance phenotypes of mlsb based on the studied genes ( meca , erma , ermc and msra ) r * : resistant phenotype ; hd : hazy d zone phenotype ; d : d phenotype ; d : d phenotype ; n : negative phenotype . among erythromycin
resistant isolates , 40 isolates had constitutive resistance r phenotype and 32 of these isolates contained both erma and ermc genes simultaneously with or without msra gene . in this study
only one isolate showed constitutive resistance hd phenotype which contained both erma and ermc genes in the absence of msra gene .
the d - zone phenotype was observed in 11 isolates showing d - shaped clear zone around clindamycin disk .
1 ) revealed that five d phenotype isolates possess erma gene alone and three isolates with d phenotype just harbored ermc gene .
one negative phenotype isolate which showed resistance to erythromycin but was sensitive to clindamycin with a clear zone around clindamycin disk did not carry erma , ermc or msra genes ( table 2 ) .
resistant phenotype isolates , 38 isolates were mrsa whereas 10 out of 15 imlsb resistant phenotype isolates were mrsa .
lane m , 1000 bp dna ladder ; lane 1 , positive control pcr for erma ( 421 bp ) and ermc ( 571bp ) genes ; lanes 23 , positive control pcrs for msra ( 940 bp ) and meca ( 310 bp ) genes ; lane 4 , negative control pcr ; lane 5 , erma and ermc positive mrsa ( a r phenotype ) ; lane 6 , erma gene positive mrsa ( a d pheno - type ) ; lane 7 , ermc gene positive mrsa ( a d phenotype ) ; lane 8 , msra gene positive mrsa ( a r phenotype ) ; lane 9 , mrsa positive ( a n phenotype ) . in this study ,
most of the isolated s. aureus strains carried both of the erma and ermc genes and a significant association was observed between different resistance phenotypes and erm genes ( p 0.01 ) .
resistance to antimicrobial agents is an important problem in clinical issues and mrsa is now one of the most common nosocomial pathogens in many countries ( 18 ) . in this study
the rate of methicillin resistance among clinical isolates of s. aureus was 57.4% which is higher than reports from tehran ( 29.752% ) ( 1922 ) , other cities in the country ( 44.456.8% ) ( 2326 ) or other countries including pakistan ( 48% ) , australia ( 33.6% ) and turkey ( 25.9% ) ( 2729 ) . on the other hand , it was lower than reports from china ( 72.8% ) and india ( 59.3% ) ( 30 , 31 ) .
geographic variations in the prevalence rate of mrsa among iranian s. aureus isolates and variations from one hospital to the others may be due to the various factors such as efficacy of practices in controlling the infection , healthcare facilities and antibiotic usages that vary in the hospitals ( 32 ) . due to the changing pattern of antibiotic resistance among s. aureus isolates , it would be wise to have periodical surveillance of these changes every 3 to 4 years ( 33 ) . in
the present study , erythromycin resistance ( 56.4% ) was higher than published reports from belgium ( 37.4% ) ( 7 ) , iran ( 42% ) ( 21 ) or india ( 51.7% ) ( 34 ) and lower than turkish ( 60.4% ) ( 29 ) or korean reports ( 77.5% ) ( 35 ) . in this study ,
the rates of constitutive , inducible and ms phenotypes among 57 erythromycin resistant isolates were 71.9 , 26.3 and 1.7% , respectively
. the observed higher rate of cmlsb than imlsb resistance phenotype among erythromycin resistant s. aureus isolates was in accordance to the other reports ( 11 , 36 , 37 ) .
rate of inducible phenotype in the erythromycin resistant s. aureus isolates was higher than other iranian studies ( 6.4% , 14% ) ( 11 , 23 ) and lower than studies from all over the world ( 6 , 34 , 37 ) . in this study ,
the ms resistance phenotype was slightly higher than what reported by another study from iran ( 11 ) and lower than other studies from greece and the us ( 6 , 13 ) . increased rate of inducible resistant isolates is attributed to the increased usages of macrolides and clindamycin .
it is crucial to perform the d - test to determine the erythromycin resistance ( 3 ) . in our study 57.8% of erythromycin
resistant s. aureus isolates had a coexistence of erma and ermc genes which was higher than the other one conducted study from iran ( 48.4% ) ( 11 ) and two published studies from turkey ( 37.5 and 18.6% ) ( 38 , 39 ) but lower than greece ( 0.5% ) and belgium ( 3% ) ( 6 , 7 ) .
msra gene was detected in 3.5% of erythromycin resistant s. aureus isolates which was higher than reports from canada ( 1% ) ( 40 ) and lower than other reports from belgium hospitals ( 5% ) ( 7 ) and turkey ( 9.5% ) ( 29 ) . coexistence of erma and ermc genes in constitutive resistance phenotype was similar to the reports in turkey ( 38 , 39 ) .
we found 10 mrsa isolates having inducible phenotype among which 5 isolates harbored erma and 3 other isolates only consisted ermc gene .
it is notable that the former group had d phenotype while the latter ones were d similar to the findings of the study performed by stward et al . ( 13 ) . in our study msra gene
was seen in 2 constitutive resistant mrsa isolates in contrast with the study published by spilopoulou et al .
it appears that the high prevalence of inducible resistance in this study may be due to the variable use of erythromycin and clindamycin in iran .
this is the first report from iran that shows differentiation of mlsb resistance phenotypes according to their corresponding genes which can provide information to help characterization of isolates for epidemiologic studies in the communities .
d - test should be an obligatory test in routine disk diffusion methods to detect inducible antibiotic resistance in treatment of infections .
on the other hand , increasing of ( erma + ermc ) and msra genes in the mrsa emphasizes on the accurate use of these antibiotics to prevent any treatment failure . | background and objectives : macrolide , lincosamide and streptogramin type b ( mlsb ) antibiotics are important in the treatment of staphylococcus aureus infections and existence of isolates with ability to resist against mlsb antibiotics is worrisome.materials and methods : in this cross sectional study , 101 s. aureus isolates were collected from patients of five selected hospitals in tehran over a period of five months .
disk diffusion tests and differentiation between constitutive and inducible resistances were carried out by d - test .
the presence of meca , msra , erma and ermc genes were detected using pcr or multiplex pcr.results:out of 101 s. aureus isolates , 58 ( 57.4% ) were methicillin resistant and 57 ( 56.4% ) expressed resistance to erythromycin .
the prevalence of constitutive mlsb ( cmlsb ) , inducible mlsb ( imlsb ) and ms ( negative ) phenotype in all erythromycin resistant isolates were 71.9 , 26.3 and 1.7% , respectively .
out of all the erythromycin resistant isolates , 57.8% harbored both erma and ermc genes which possessed constitutive resistance .
8.7% of the isolates contained erma gene alone which possessed inducible resistance with d phenotype and 5.2% of isolates just contained ermc gene which had inducible resistance with d+ phenotype .
msra gene was detected in 3.5% of the erythromycin resistant s. aureus isolates with constitutive resistance .
none of the genes were detected among ms phenotypes.conclusion:in this study , most of s. aureus isolates carried both erma and ermc genes and there was a significant relationship ( p value 0.05 ) between different resistance phenotypes and erm genes . |
in complex disorders where variations in more than one gene are expected to contribute to disease risk , researchers often hypothesise that particular groups of genes or genomic locations are enriched with true disease - susceptibility genes , based on various lines of evidence . for example , groups of genes found to be differentially expressed in a case - controlled microarray experiment or the human loci syntenic to those identified by linkage in a mouse disease model are likely to be enriched in susceptibility genes .
it can be suggested , for example , that the genes involved in glutamate neurotransmission are enriched with schizophrenia - susceptibility genes , or -- combining more than one line of evidence -- that differentially expressed glutaminergic genes in particular are likely to be enriched .
researchers may wish to corroborate such hypotheses by testing whether the members of an identified group of genes are located in areas showing evidence of genetic linkage to the disease of interest .
we propose a method for using genome - wide linkage data and the widely used non - parametric linkage ( npl ) score to test for the enrichment of groups of genes or genetic locations in disease - susceptibility genes .
the npl score is designed to have , at any locus , a standard normal distribution with a mean of 0 and a standard deviation of 1 under the null hypothesis of no linkage .
this means that , although for any given unlinked locus the expectation is an npl score of 0 , stochastic variation creates scores that can take positive or negative values . under the alternative hypothesis of linkage , stochastic variation at the true disease loci is still present , but the expectation is at a value higher than 0 .
the magnitude of the expected value depends on the sample size , the available genetic information and the effect size of the locus . for most complex diseases
, it is assumed that individual risk loci will have small effects . as a result ,
the superimposed stochastic variation can mask some truly linked loci or create signals where no true linkage is present , both situations leading to errors and/or failed replication studies for true disease loci . if one could have a priori knowledge of the true disease loci , one could achieve greater significance by studying the group of loci in concert because of the consistent trend for increased scores , even in the absence of significant scores at each one of the individual loci . as the number of true loci examined together rises , the noise from the underlying stochastic variation will asymptotically approach 0 and their average npl score will asymptotically approach the average of their individual expectations , which is greater than 0 . by contrast , for unlinked loci , the individual expectation is 0 ; as the number of unlinked loci examined in concert increases , their average npl will asymptotically approach 0 . based on these properties of the npl score
, we can use linkage analysis data to test whether a pre - defined group of loci is enriched for true disease - linked loci .
this can be done by calculating the average npl score of the group of loci and comparing it against a null distribution of average scores derived by randomly drawing groups of loci of equal size .
the null hypothesis is that the proportion of true linked loci among the group of loci tested is not different from the proportion expected when choosing random loci , while the alternative hypothesis is that the proportion is greater , and hence the group is enriched .
( note that , as defined here , the proportion of true loci in the group tested corresponds to 1 minus the false discovery rate of the group . )
we assessed the power of our proposed method through simulations using a variety of disease models and varying the number of errors in location predictions .
we showed that this method can be powerful , even when less than half of the examined locations are real disease loci .
we must note here that we used the npl score because it is commonly available and because its statistical properties make it easier to present our hypothesis .
since significance is determined via permutations and no distribution is assumed , however , the method is applicable to any statistic . also , we are aware that other summary statistics , such as the product of p - values ( more often used to show the presence of at least one true locus in a group ) , can serve for the same purpose .
we assumed a baseline risk for the disease of 0.9 per cent , representing non - genetic factors . we used the ' - -simulate ' function in the merlin analysis package to generate genome - wide marker data for nuclear families ( two parents and four offspring ) , including five , ten or 20 biallelic ( disease ) loci carrying risk alleles of frequency p that independently increase the risk of disease two- or threefold ( this corresponds to their relative risk ) .
the disease allele frequency p was equal for all loci and was empirically adjusted to provide a population prevalence of 3 per cent .
this corresponds to 70 per cent heritability ( genetic / total variance ) , consistent with reports for many complex disorders . after generating data for a large number of families ( up to 200,000 -- or 1.2 million individuals ) ,
the genotypes at the disease loci for each individual were examined , the risk was determined based on those genotypes and disease status was assigned with a probability corresponding to the risk .
for example , a person carrying four risk alleles with a relative risk of 2 had a probability of 2 0.009 = 0.144 of being affected .
sufficient families were generated every time to ascertain 1,000 sibling pairs and 60 sibling triads ( 1,180 sibling pairs in total ) or 500 sibling pairs and 30 sibling triads ( 590 sibling pairs ) .
this ratio of pairs to triads corresponds to the most efficient choice , given the observed simulated families , but it is not far from common sibling size distributions in the complex disease literature .
the genotypes of all markers , excluding the biallelic disease loci , were used for genome scans for linkage using the merlin software to calculate npl scores across the genome .
markers other than the disease loci had six equifrequent alleles , spaced 10 centimorgans ( cm ) apart , and there were no missing data .
a total of 359 microsatellite markers were simulated , starting on each chromosome at genetic position 0 and placing one marker every 10 cm until the end of the chromosome ; therefore , a genetic length of 0 to less than 10 cm was at the telomere of each chromosome .
although this might be a slightly large family size compared with today 's average in the western world , it is less so for families that are ascertained today through their adult affected offspring .
this family size was also necessary to make the generation of enough pedigrees for ascertainment computationally feasible .
for each simulated scan , the location of the disease loci varied and these were placed randomly in the genome , allowing for co - localisation of more than one disease locus and for zero distance with scan markers if it so happened by chance . in order to assess the power of our method under many possible scenarios , we studied multiple genome scans under multiple disease parameters including : 1 ) the number of disease genes was set to five , ten or 20 ; 2 ) the increase in risk from each risk allele was set to 2 or 3 relative to the baseline risk ; 3 ) the number of ascertained families was 500 pairs + 30 triplets or 1,000 pairs + 60 triplets .
for each of the 12 possible sets of parameters , 25 genome scans were generated .
for each genome scan , we examined a number of different scenarios regarding the number of true and non - true disease loci in the group to be tested .
when some non - true locations , or not all the real locations , were included , the groups were chosen 100 times at random to account for the stochastic variation inherent to the selection . in order to determine significance for each of the 100 selected groups ,
the average npl of each was compared with the null distribution formed by the average scores of 1,000 random groups of equal size -- that is , groups chosen without taking into account whether or not the included loci correspond to disease gene locations .
the 2,500 empirical significance values obtained from 25 scans 100 group permutations were used to determine the power for each model ( each cell on table 1 ) . since in these simulated data the alternative hypothesis ( as stated above ) is always true , the number of times that the empirical significance is less than the desired significance level , , corresponds to the power .
the power of our method for different simulated models ( five , ten or 20 disease loci , 1,180 or 590 sibling pairs , relative risk ( rr ) of 2 or 3 ) different levels of enrichment for true loci and different levels of significance .
we assumed a baseline risk for the disease of 0.9 per cent , representing non - genetic factors . we used the ' - -simulate ' function in the merlin analysis package to generate genome - wide marker data for nuclear families ( two parents and four offspring ) , including five , ten or 20 biallelic ( disease ) loci carrying risk alleles of frequency p that independently increase the risk of disease two- or threefold ( this corresponds to their relative risk ) .
the disease allele frequency p was equal for all loci and was empirically adjusted to provide a population prevalence of 3 per cent .
this corresponds to 70 per cent heritability ( genetic / total variance ) , consistent with reports for many complex disorders . after generating data for a large number of families ( up to 200,000 -- or 1.2 million individuals ) ,
the genotypes at the disease loci for each individual were examined , the risk was determined based on those genotypes and disease status was assigned with a probability corresponding to the risk .
for example , a person carrying four risk alleles with a relative risk of 2 had a probability of 2 0.009 = 0.144 of being affected .
sufficient families were generated every time to ascertain 1,000 sibling pairs and 60 sibling triads ( 1,180 sibling pairs in total ) or 500 sibling pairs and 30 sibling triads ( 590 sibling pairs ) .
this ratio of pairs to triads corresponds to the most efficient choice , given the observed simulated families , but it is not far from common sibling size distributions in the complex disease literature .
the genotypes of all markers , excluding the biallelic disease loci , were used for genome scans for linkage using the merlin software to calculate npl scores across the genome .
markers other than the disease loci had six equifrequent alleles , spaced 10 centimorgans ( cm ) apart , and there were no missing data .
a total of 359 microsatellite markers were simulated , starting on each chromosome at genetic position 0 and placing one marker every 10 cm until the end of the chromosome ; therefore , a genetic length of 0 to less than 10 cm was at the telomere of each chromosome .
although this might be a slightly large family size compared with today 's average in the western world , it is less so for families that are ascertained today through their adult affected offspring .
this family size was also necessary to make the generation of enough pedigrees for ascertainment computationally feasible .
for each simulated scan , the location of the disease loci varied and these were placed randomly in the genome , allowing for co - localisation of more than one disease locus and for zero distance with scan markers if it so happened by chance .
in order to assess the power of our method under many possible scenarios , we studied multiple genome scans under multiple disease parameters including : 1 ) the number of disease genes was set to five , ten or 20 ; 2 ) the increase in risk from each risk allele was set to 2 or 3 relative to the baseline risk ; 3 ) the number of ascertained families was 500 pairs + 30 triplets or 1,000 pairs + 60 triplets .
for each of the 12 possible sets of parameters , 25 genome scans were generated .
for each genome scan , we examined a number of different scenarios regarding the number of true and non - true disease loci in the group to be tested .
when some non - true locations , or not all the real locations , were included , the groups were chosen 100 times at random to account for the stochastic variation inherent to the selection . in order to determine significance for each of the 100 selected groups ,
the average npl of each was compared with the null distribution formed by the average scores of 1,000 random groups of equal size -- that is , groups chosen without taking into account whether or not the included loci correspond to disease gene locations .
the 2,500 empirical significance values obtained from 25 scans 100 group permutations were used to determine the power for each model ( each cell on table 1 ) . since in these simulated data the alternative hypothesis ( as stated above ) is always true , the number of times that the empirical significance is less than the desired significance level , , corresponds to the power .
the power of our method for different simulated models ( five , ten or 20 disease loci , 1,180 or 590 sibling pairs , relative risk ( rr ) of 2 or 3 ) different levels of enrichment for true loci and different levels of significance .
table 2 summarises our general observations from the simulated genome scans with 1,180 affected sibling pairs .
a linkage peak was considered to contain a real disease locus if the npl scores between the original location of the binary disease marker and the observed peak did not drop by more than one unit less than the score at the peak .
even with five loci of relative risk 3 , the top npl score did not reach genome - wide significance on most scans ( 25 scan average = 4.22 ) , according to the criteria proposed by lander and kruglyak ( for the genome - wide significant p = 2.2 10 , an npl score of 4.4 is required ) , in accordance to what has been observed in real data analyses and predicted by risch and merikangas .
it is notable and encouraging , however , that , across the models we tested , 40 - 92 per cent of scans showed strongest linkage at a real locus .
we also observed that even when there are only five true loci on average , one of these loci is not among the top ten peaks of a scan and would therefore not be detected .
again , this observation is very much in agreement with the experience from linkage studies of complex disorders , as many linkage findings that have been considered to carry strong evidence have often not been replicated in subsequent studies of different pedigrees .
as expected , the npl scores and the fraction of true positives among the top linkage peaks decrease as the number of disease loci increases and as their relative risk decreases .
an increase in the fraction of true findings is counter - intuitively observed as the number of true disease loci and the number of top linkage peaks examined increases to 20 ; however , this does not correspond to an increase in the fraction of real genes identified . when looking at table 2 , the reader should keep in mind that when there are only five real genes and a set of 20 loci is tested , the maximum possible fraction of true loci in the set is 5/20 = 25 per cent .
overall , our observations confirm that our confidence in the linkage peaks of a single scan should be somewhat reserved until we observe replication , but also that non - replication of a linkage finding does not necessarily discredit a positive finding . in other words , it will take more than a few linkage scans to develop strong confidence in the location of true susceptibility loci for a complex disorder
. results of simulated scans with 1,180 sibling pairs regarding their success in identifying the disease gene locations .
simulation parameters : h = heritability , k = prevalence , rr = relative risk for each risk allele . the number of simulated disease loci is shown .
for the one , three , five , ten and 20 top scoring loci for each of the 25 simulated genome scans , we show how many coincided with true disease loci ( and their percentages ) , as well as their average non - parametric linkage ( npl ) score .
table 1 presents an evaluation of the power of the approach we propose here for examining multiple genomic locations for linkage using a summary statistic , namely the average npl score .
in particular , it shows the power to detect enrichment by examining the average npl score at levels of = 0.05 and 0.01 and for different disease models calculated through our computer simulations . for a 1,180 sibling pair scan , and at the nominal level of = 0.05 , it is of interest
that for a relative risk of 3 and for as many as ten disease loci , we can observe significance with power > 80 per cent even if only one - third of the locations in the group are true .
for a relative risk of 2 , we have 80 per cent power if half of the locations in the group are true . for 20 segregating loci and a relative risk of 3
, we can only tolerate ten non - real locations in a group that includes all 20 correct locations if we wish to have 80 per cent power .
figure 1 provides a three - dimensional graph showing how power increases when there are fewer real loci contributing to the risk and when more of these are included in the group .
as the group gets larger and the fraction of true loci that are included is reduced , however , the power decreases .
power of our method when using a linkage scan of 1,180 sibling pairs and five ( a ) , ten ( b ) or 20 ( c ) loci with a relative risk of 3 .
x - axis : number of real loci included in the tested group ; y - axis : fraction of real loci in the group ; z - axis : power to detect the enrichment . based on our observations on the true positive enrichment of the top linkage peaks ( table 2 ) and on the power of averaging npl scores ( table 1 )
our simulations indicate that , as expected , the group of top findings of a linkage scan is enriched in true disease locations .
therefore , provided that there are not too many true loci with too small effects , when this group of top linkage peaks is tested against an independent linkage scan , it should show a significantly elevated average npl score .
we used our genome scan data on late - onset alzheimer 's disease ( load ) to test this hypothesis .
this scan was peformed on a previously described collection of pedigrees from the national institute of mental health genetics initiative , for which we have previously reported genome scan results .
the one gene known to be involved in load is apoe , and its behaviour in terms of risk is very similar to our simulated models . it has been suggested that another 4 - 5 loci with effects similar to apoe may be involved in load . adopting a study design that enabled us to keep most variables equal and
yet have two independent scans , we sorted the pedigrees by their assigned identification numbers ( signifying collection site and collection sequence ) and split them at the point that gives two sample sets ( sets a and b ) of equal numbers of sibling pairs ( 296 pairs each ) .
we then ran a genome - wide linkage analysis on both sets , ranked the top scoring 30 locations from scan a , selected groups of five , ten , 15 ... 30 locations starting from the top and tested their average npl in scan b. we note that splitting the data has no benefit for gene discovery , but we did this here as an exercise to show proof of principle because it provided us with a hypothesis that we could readily test using our method , namely that the peaks of a genome scan are enriched in true loci . given the small sample sizes ( 296 sibling pairs per scan ) , our power might have been low , since the underlying model is unknown ; however , we viewed this analysis as exploratory .
table 3 shows the empirical significance obtained by selecting the best five and up to 30 locations from the top linkage peaks based on scan a , and testing their average npl against the data from scan b. although the mean npl of the five top locations was not significantly high , once the number was raised to ten and 15 , the scores were significant , suggesting enrichment in disease gene locations .
we consider that this not only validates our method but that it is also very encouraging regarding the validity of the findings of our genome scan , suggesting that the top linkage peaks are indeed enriched in real disease loci to a significant degree .
although some might consider this notion to be obvious , it is contingent on the underlying disease model and might not necessarily be true . based on the observations from the simulated linkage scans , and the expected low power of this test on 296 sibling pairs
, this also suggests that the number of substantial disease loci is not too great and that their relative risks are not too small . as we performed comparisons in six groups , we next wanted to see if our findings were significant at the study - wide level .
the strong correlation between the tested groups makes bonferroni correction too conservative , so we tested this empirically .
we chose 10,000 groups of 30 loci and tested inclusive subgroups of five , ten , 15 ... 30 members , as we did with the real data against the scan b results .
a p - value of 0.016 or smaller in any sub - group was obtained 606 times , providing a study - wide significance of 0.06 .
two sets of pedigrees were used for scans a and b. groups of top linkage peaks from scan a ( their size is shown in column 1 ) were then tested for enrichment on the results of scan b. column 2 shows the empirical p - values for these groups .
we have shown how one can test for the enrichment of a group of genomic locations for disease loci using linkage genome scan data .
for example , one can compare the results of two independent genome scans , as described here for alzheimer 's disease , in the same or different organisms .
alternatively , one can test prior results of expression studies or genome - wide association analyses , or genes belonging to specific pathways or interacting with a suspected disease gene .
the method could be extended to applying weights to individual locations based on the strength of prior evidence .
this is highly intuitive for testing locations that carry a score or a significance value ( such as linkage , expression or association results ) but less so for other types of groups ( interacting proteins , members of a functional group , etc ) .
one could also extend the approach by testing groups on data other than linkage results , yet such approaches require further method development because there can be a number of issues that need to be addressed .
the use of sum statistics for snp association data has been described previously by wille et al . , hoh et al . and kim et al .
these authors sought methods to test for associations in multilocus disorders , with the notion of increasing power to detect associations with any one of the loci by examining groups of snps or other dna markers in concert .
by contrast , we sought to develop a method specifically for testing the hypothesis that a group of pre - selected genomic locations is enriched for disease loci .
our method is suitable for testing any group of genes or locations on pre - existing data .
in fact , our method could complement and add to the validity of the findings from other snp set association studies .
it is necessary to make sure that the linkage data used for testing the enrichment hypothesis were not in any way used to generate the hypothesis .
for example , if one tests genes that have been reported to be associated with a disease , it is necessary to use linkage data generated and/or published after the associations , as there is a strong bias towards association testing in linked regions .
if the linkage data were known before the association studies , the genes might have been examined because of the positive linkage scores and testing their scores on the same linkage scan is certain to give a false positive result .
for example , there are numerous association studies on alzheimer 's disease and we could have used our linkage data to test whether the group of positive findings is enriched for true genes .
the pedigrees used in our study , however , have been publicly available and used for genome scans since 1999 . many association studies that followed were biased towards examining linked regions and
we would need results from an unbiased genome screen for association to perform a valid test for enrichment .
one also needs to consider that although the power of the method is substantial , it will quickly diminish if multiple hypotheses of little merit are examined , as this will require substantial correction for multiple comparisons . additionally ,
as the true underlying disease model is not known , negative results can not be taken as evidence against a hypothesis and must be interpreted with caution .
although failing to reject the null hypothesis might suggest that the alternative is wrong , it might also be due to decreased power resulting from the small effect of individual genes , the large number of genes involved , insufficient enrichment of the selected locations in true disease genes or the small number of pedigrees in the linkage study .
regarding the last point , the approach could be extended to simultaneous examination of two or more linkage scans to increase power without the need to combine the genotype data with all the inherent difficulties of doing so .
one can simply perform permutations of the same group of random loci on both scans and examine the distribution of the combined average npl score against the observed average of the two scans for the tested group .
our simulation data can provide some guidance on the optimal selection of group size . as figure 1 shows , when less than 50 per cent of the loci in the group are real , the power starts to diminish .
significant loss of power is also observed when less than half of all true loci are included in the group ( table 1 ) ; thus , we suggest using the maximum group size that does not exceed twice the predicted number of true loci . our data on load support this , as the predicted number of loci conferring a relative risk of 2 - 3 is five , and we obtained our strongest finding with a group of size of ten .
when information on an expected number of disease genes is available , we suggest avoiding multiple comparisons by defining a priori the size of the tested group to roughly twice that number . if one wishes to test multiple group sizes , correction for the multiple correlated comparisons is required using empirical methods . as we observed in our example in alzheimer 's disease , the predicted group of ten loci would have provided the highest significance , while testing six groups resulted in a study - wide p - value of 0.06 .
variations in group size can be useful in determining the most enriched group , yet it might be best to perform this analysis after significance has been established .
our example using alzheimer 's disease linkage data showed how positive findings can not only confirm a hypothesis -- in this case , confirm that a significant proportion of disease loci are amidst the top linkage findings -- but also lead to insight regarding the possible underlying model .
it has been previously proposed that about five loci , each conferring a relative risk of 2 - 3 for load , segregate in the population . according to table 2 , for 1,180 sibling pairs and five loci with a relative risk of 3
, we would expect that 3.2 of the top five and four of the top ten linkage peaks would be real .
if we compared these against a linkage scan of 590 sibling pairs , extrapolating from table 1 , we would expect to have somewhat more than 80 per cent power to detect this degree of enrichment .
although our sample for both genome scans was about half the size of this sample and presumably had significantly less power , we detected the enrichment in our data . having a positive finding in this analysis that is consistent with the proposed number of loci and relative risks is very encouraging , as it suggests that linkage analysis has pointed to some truly linked regions in our alzheimer 's genome scan .
the analytical approach we propose here is simple and , since it calculates the significance of findings based on permutations , robust to type i errors , provided that the prediction of the genomic locations to be grouped and analysed is in no way biased by the linkage data on which the test will be performed .
we showed that the approach has substantial power under disease models with a moderate number of risk genes and moderate relative risks .
we believe that as more and more diverse data accumulate through the various high - throughput technologies , it is increasingly important to devise more methods of combining and crossvalidating the resulting information that will help us succeed in our effort to understand complex disorders .
( ag021804 ) , and by an award from the neurosciences education and research foundation to d.a . | genes for complex disorders have proven hard to find using linkage analysis .
the results rarely reach the desired level of significance and researchers often have failed to replicate positive findings .
there is , however , a wealth of information from other scientific approaches which enables the formation of hypotheses on groups of genes or genomic regions likely to be enriched in disease loci .
examples include genes belonging to specific pathways or producing proteins interacting with known risk factors , genes that show altered expression levels in patients or even the group of top scoring locations in a linkage study .
we show here that this hypothesis of enrichment for disease loci can be tested using genome - wide linkage data , provided that these data are independent from the data used to generate the hypothesis .
our method is based on the fact that non - parametric linkage analyses are expected to show increased scores at each one of the disease loci , although this increase might not rise above the noise of stochastic variation . by using a summary statistic and calculating its empirical significance ,
we show that enrichment hypotheses can be tested with power higher than the power of the linkage scan data to identify individual loci . via simulated linkage scans for a number of different models , we gain insight in the interpretation of genome scan results and test the power of our proposed method .
we present an application of the method to real data from a late - onset alzheimer 's disease linkage scan as a proof of principle . |
the neurotransmitter pharmacology of nociceptive processing is of great importance in our understanding of pain mechanisms and therapy .
understanding of the neurotransmitters and receptors involved in nociception is extensive at the level of the peripheral nervous system and the spinal cord ( lesage , 2004 ; millan , 1999 ) .
the ventrobasal thalamus ( vb ) is a pivotal processing point for the integration of somatosensory information ascending from the spinal cord with a prominent descending cortico - thalamic input from layer 6 of the corresponding somatosensory cortex ( sherman , 2012 ) .
previous work from this laboratory and others has shown that responses of thalamic neurones to noxious peripheral stimuli are largely mediated by glutamate receptors , in particular the ionotropic nmda receptor ( bordi and quartaroli , 2000 ; eaton and salt , 1990 ; kolhekar et al . ,
1997 ) and the metabotropic glutamate ( mglu ) receptors , mglu1 and mglu5 ( eaton et al . , 1993 ; salt and binns , 2000 ; salt and turner , 1998 ) .
there are eight mglu receptor subtypes ( mglu1-mglu8 ) that can be placed into three groups ( group i , ii , iii ) based on sequence homology , intracellular transduction cascade , and agonist / antagonist pharmacology ( niswender and conn , 2010 ) .
group i receptors ( mglu1 , mglu5 ) are often ( but not exclusively ) localised post - synaptically where they may couple to inositol phosphate metabolism and enhance post - synaptic excitability via changes in k conductances and/or modulation of ionotropic glutamate receptors ( niswender and conn , 2010 ) .
in addition to selective agonists and antagonists , a novel class of pharmacological agents acting at mglu receptors has more recently become available , the positive allosteric modulators ( pams ) ( nicoletti et al . , 2011 ) .
pams act at sites on the receptor distinct from the orthosteric ligand ( glutamate ) binding site and enhance the activity of receptors in response to orthosteric agonists ( nicoletti et al . , 2011 ) .
this has advantages in that pams can be highly selective for single mglu receptor subtypes and can function to potentiate the physiological activation of receptors by endogenous glutamate .
we have shown that one such pam , ro67 - 4853 ( knoflach et al . , 2001 ) , can be used in in vivo experiments to potentiate responses mediated via mglu1 receptors in an activity - dependent manner ( salt et al . , 2012 ) .
thus , ro67 - 4853 is an appropriate tool to investigate possible mglu1 involvement in thalamic nociceptive processing . the present study aimed to investigate the involvement and function of mglu1 receptors in nociceptive processing in the thalamus .
we have achieved this by potentiating mglu1 receptor mediated responses with the selective pam ro67 - 4853 and by reducing mglu1 receptor activation using the selective antagonist ly367385 ( clark et al . , 1997 ) whilst recording nociceptive responses of thalamic neurones .
furthermore , given the association of mglu1 receptors with cortico - thalamic pathways ( godwin et al .
, 1996 ; martin et al . , 1992 ; vidnyanszky et al . ,
1996 ) , we investigated the effect of somatosensory cortex inactivation on nociceptive responses of thalamic neurones . we show that it is possible to substantially change nociceptive responses of thalamic neurones by increasing or decreasing the degree of mglu1 receptor activation and that nociceptive responses of thalamic neurones are dependent upon a functional cortico - thalamic projection .
this is important in understanding sensory processing and the design of novel analgesic therapies , and underlines the critical role of mglu1 receptors in sensory processing in the thalamus .
experiments were carried out in male adult wistar rats ( 270400 g ) anaesthetised with urethane ( 1.2 g / kg , i.p . ) , as detailed previously ( salt , 1987 ; salt and binns , 2000 ; salt et al . , 2012 ) .
animals were purchased from harlan ( uk ) and were housed on a 12 h light / dark cycle with unlimited access to food and water .
all procedures were subject to local ethical committee review , were approved by the home office ( uk ) and were in accordance with the animals ( scientific procedures ) act 1986 .
an approximately 5 mm - square unilateral craniotomy centred over the thalamus ( 3 mm lateral to the midline , 5 mm rostral to the inter - aural line ( paxinos and watson , 1988 ) ) was made and the dura resected to expose the surface of the cortex .
in addition to overlying the thalamus , this area of cortex contains the hindlimb and trunk / tail representation of the s1 somatosensory cortex ( chapin and lin , 1984 ) . in some experiments
the craniotomy was surrounded by a small open chamber cemented to the skull that could be filled with either mineral oil or physiological saline ; in the remaining experiments the surface of the brain was protected by agar ( 2% in physiological saline ) .
extracellular recordings were made from single neurones in the vb and immediately dorsal thalamus using either tungsten - in - glass electrodes or , for pharmacological experiments , the central barrel of seven - barrel glass iontophoretic electrodes .
single neurone action potential spikes were gated using a hardware spike - discriminator whose output pulses were timed and recorded by a ced1401 interface and computer system with spike2 software . the amplitude and shape of the gated action potentials
neurones were identified on the basis of their stereotaxic location ( ap + 5.0 mm from lambda , lateral 2.9 mm from midline , depth 4.65.2 mm from surface ) and their responses to somatosensory ( nociceptive and non - nociceptive ) stimuli , as described previously ( guilbaud et al . , 1980 ; peschanski et al . , 1980 , 1983 ; salt and binns , 2000 ) .
nociceptive responses were evoked by immersion of part of either the contralateral hindpaw or the tail in water of 52c for 20 s. responses to such stimuli were typically increases in action potential firing during the course of the stimulus and outlasting the stimulus by up to 2 min , as described previously ( peschanski et al . , 1980 ) .
similar response profiles were observed irrespective of the recording electrode type or the type of craniotomy preparation .
noxious stimuli were repeated at regular 5-min intervals in experiments where modulation of nociceptive responses was investigated ( see below ) . for pharmacological experiments , substances under investigation
were applied in the recording location from the six outer barrels of the electrode using the iontophoretic technique ( stone , 1985 ) with a neurophore bh2 system .
each of the outer barrels contained a selection from one of the following substances : nmda ( n - methyl - d - aspartate , 50 mm , ph 8.0 in 150 mm nacl ) ; ampa ( s - alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionate , 50 mm , ph 8.0 in 150 mm nacl ) ; ly367385 ( 100 mm in water , ph 8.0 ) ; dhpg ( s-3,5-dihydroxy - phenylglycine , 50 mm in 150 mm nacl , ph 3.5 ) ; ro67 - 4853 ( 2 mm in 10% dmso in 150 mm nacl , ph 7.5 ) , vehicle control ( 10% dmso in 150 mm nacl , ph 7.5 ) , pontamine sky blue dye ( 2% in 1 m nacl ) , 1 m nacl .
all pharmacological agents were obtained from tocris bioscience ( uk ) , apart from ro67 - 4853 , which was a gift from roche pharmaceuticals ( switzerland ) .
agents were prevented from diffusing from the iontophoresis barrel by applying a retaining current ( 515 na ) of opposite polarity to the ejection current .
automatic current balancing was routinely performed through the 1 m nacl - containing barrel . in experiments where the effects of either the pam or antagonist on agonist responses were evaluated ,
regular repeated cycles of agonist ejections were set up and initiated by the computer system which also produced online peristimulus - histograms ( psths ) of single - neurone activity .
the effects of the pam or antagonist on agonist effects were assessed by continuous concurrent iontophoretic application of these agents during several cycles of agonist ejection .
thus , although it is not possible to give absolute tissue concentrations of agents , we are confident that the doses used are within the range of producing physiologically relevant and pharmacologically selective effects . in experiments where the effects of either the pam or antagonist on nociceptive responses were investigated , these agents were ejected with similar iontophoretic parameters to those found to be effective on responses to agonists . in experiments designed to investigate the influence of cortical activity on thalamic responses , we inactivated the s1 somatosensory cortex by cooling ( clemo and stein , 1986 ; diamond et al . , 1992 ) .
after responses to noxious stimuli of a neurone were established , the warm physiological saline in the well overlying the cortex was gently aspirated and replaced with chilled ( 4c ) saline every 23 min for up to 10 min and the effect on the nociceptive responses noted . finally , the saline was replaced with warm saline and nociceptive responses were further recorded . in separate experiments ,
a miniature thermocouple was inserted into the cortex to a depth of 1 mm and it was found that the cooling procedure reduced the temperature at this point to 15c . when the thermocouple was moved down into the body of the thalamus , no change in temperature could be detected when the cortical cooling procedure was performed .
responses to agonists or noxious stimuli were quantified as the number of action potentials evoked by agonist ejection or stimulus , from which psths were plotted .
the effects of the pam , the antagonist , or cortical inactivation on these responses were assessed by calculating the agonist or stimulus response during these experimental manipulations as a percentage of the response under control conditions . in the case of nociceptive responses ,
maintained response components were computed : the initial component was the number of action potential spikes evoked during the nociceptive stimulus whereas the maintained response was the number of action potential spikes occurring during the minute immediately after the stimulus .
data from individual neurones were used to compute mean values of effects ( s.e.m . ) .
statistical comparisons of these values under control conditions and during experimental manipulations were made using the wilcoxon signed rank test .
recordings were made from 25 neurones that were characterised as nociceptive thalamic neurones on the basis of their stereotaxic location and responses to noxious stimuli directed at the contralateral limbs and tail as described previously ( guilbaud et al .
typically these neurones were located above or lateral to the vibrissal representation in the vb complex , had low spontaneous firing rates ( 0.052.4 spikes per second ) and responded to noxious stimuli with a graded increase in firing rate that outlasted the stimulus , as described previously ( eaton and salt , 1990 ; guilbaud et al . , 1980 ; peschanski et al . , 1980 ; salt and binns , 2000 ) .
i agonist dhpg ( 2080 na , 1020 s ) caused increases in action potential firing rate of the thalamic neurones , as previously described ( salt and binns , 2000 ; salt et al . , 2012 ) .
previous work from this laboratory has indicated that this excitatory response to dhpg is mediated predominantly via mglu1 receptors ( salt and binns , 2000 ) . in order to evaluate the ability of ro67 - 4853 to potentiate mglu1
receptor mediated responses of nociceptive thalamic neurones , we co - applied this pam ( 50150 na ) during regular ejections of dhpg .
we found that this potentiated the dhpg responses to 282 87% ( n = 6 , p < 0.05 ) of their pre - ro67 - 4853 control values on nociceptive neurones ( fig . 1 ) .
on vibrissae - responsive ( non - nociceptive ) neurones a similar effect ( 267 33% of control , n = 19 , p <
this effect was typically reversible within 10 min of the termination of the ro67 - 4853 application .
it appeared that the greatest degree of potentiation could be achieved when the control response to dhpg was relatively low in terms of increased firing of action potentials .
this was borne out by the apparent inverse correlation between the potentiating effect of ro67 - 4853 and the magnitude of the control response to dhpg ( p < 0.001 ) ( fig .
these findings indicate that ro67 - 4853 can potentiate the mglu1-receptor - mediated responses to dhpg of thalamic nociceptive neurones , in agreement with our previous findings on vibrissa - responsive thalamic neurones ( salt et al . , 2012 ) .
when ro67 - 4853 was applied during noxious stimulation on 6 thalamic neurones , the nociceptive responses were potentiated to an overall 247 99% of control values ( p < 0.05 ) , with a more pronounced effect on the latter ( maintained ) components of the nociceptive responses than on the initial component in 5 of the 6 neurones ( figs . 2 and 3a ) .
the effect of ro67 - 4853 reversed upon termination of the ejection . on 4 of these 6 neurones the responses to repeated noxious stimuli were further recorded during co - application of the mglu1 receptor antagonist ly367385 ( 2040 na ) ( fig 2 ) .
this antagonist reduced the nociceptive responses in these neurones and in a further 5 neurones where ro67 - 4853 had not been tested ly367385 had a similar effect .
overall , on the 9 neurones where nociceptive responses were studied with ly367385 , the antagonist reduced responses to 17 9% of control levels with little difference in effect between initial and maintained components of the response profiles ( fig .
3b ) . in order to verify the selectivity of ly367385 on this population of neurones , we recorded from 8 neurones that were excited by regular ejections of dhpg , nmda and ampa . on all of these neurones , co -
application of ly367385 reduced dhpg responses ( to 6 5% of control ) while having little effect on responses to either nmda or ampa ( fig .
in view of the possibility that the cortico - thalamic projection contributes to the mglu1 receptor - mediated component of thalamic nociceptive responses , we carried out a set of experiments where we inactivated somatosensory cortical activity by cooling the cortex whilst recording thalamic nociceptive responses . in these experiments
, there was a substantial reduction in the magnitude of nociceptive responses of thalamic neurones to 14 8% of control ( n = 5 ) ( fig . 5 ) .
responses returned to near - control levels in all cases once the cortical cooling procedure had been terminated .
we have previously reported that local application of the mglu1 receptor pam , ro67 - 4853 , can enhance dhpg - evoked excitations of somatosensory vibrissae - responsive vb neurones ( salt et al . , 2012 ) .
the primary finding of the present study is that this pam has similar effects on nociceptive thalamic neurones and is able to substantially enhance nociceptive responses of these neurones .
furthermore , in an extension to our previous studies , we have shown that nociceptive thalamic responses are sensitive to antagonism by a selective mglu1 receptor antagonist ( ly367385 ) , and finally that the nociceptive responses are dependent on an intact cortical input .
inactivation of the sensory cortex has been used as a means of investigating cortical influences on subcortical areas in a variety of species and sensory systems ( binns and salt , 1996 ; clemo and stein , 1986 ; marrocco et al . , 1982 ;
, 1993 , 1994 ; wickelgren and sterling , 1969 ) including the rat somatosensory cortex ( diamond et al . , 1992 ; yuan et al . , 1985 ) :
cooling of somatosensory cortex appears to be a useful means of producing a reversible inactivation to study corticofugal influences on somatosensory thalamic processing in the rat ( diamond et al . , 1992 ) .
( 1992 ) found that such a procedure had little effect on vibrissa responses of ventrobasal thalamus neurones although responses of posterior group ( pom ) thalamic neurones were reduced . in the present study , using similar procedures , we found that nociceptive responses were profoundly reduced by cortical inactivation , and this might at first reflect a greater involvement of cortico - thalamic circuitry in nociceptive responses compared to vibrissal responses but might also reflect patterns of vibrissal input ( salt et al . , 2012 ) .
ro67 - 4853 has been documented as a pam that is selective at mglu1 receptors across a range of species with an ec50 of approximately 0.1 m ( knoflach et al . , 2001 ) .
this selectivity is borne out by our present finding that ro67 - 4853 can enhance responses to dhpg in a similar manner to the potentiation seen on hippocampal neurones in vitro ( knoflach et al . , 2001 ) , and is consistent with the high levels of expression of mglu1 receptors compared to mglu5 receptors in the thalamus ( martin et al . , 1992 ; neto et al . , 2000 ; shigemoto et al . ,
our findings that nociceptive responses are potentiated by an mglu1 pam and reduced by an mglu1 antagonist provide compelling evidence to suggest that mglu1 receptors are activated during this physiological stimulation .
the most likely physiological source of the mglu1 receptor - mediated component of thalamic responses is the layer 6 cortico - thalamic input to thalamic neurones in view of the anatomical location of these receptors on distal dendrites close to terminals likely of cortical origin ( godwin et al .
, 1996 ; martin et al . , 1992 ; vidnyanszky et al . ,
this is further supported by in vitro electrophysiological experiments where stimulation of the cortical input to thalamic neurones that can be reduced with mglu1 antagonists ( reichova and sherman , 2004 ; turner and salt , 2000 ) .
consistent with this , we have found in the present study that inactivation of the somatosensory cortex reduces the nociceptive responses of thalamic neurones . taken together
, this suggests that responses of thalamic neurones to noxious stimuli are a consequence of recruitment of cortical circuitry in addition to sensory input .
it is of interest that a similar suggestion has been made for thalamic responses in the somatosensory vibrissal system and the visual system in vivo and that the degree of recruitment may depend on the stimulus parameters that are used ( rivadulla et al . , 2002 ; salt et al . , 2012 ) .
it is evident that manipulation of mglu1 activation either in an upward ( with pam ) or downward ( with antagonist ) direction has a profound effect on the nociceptive responses of thalamic neurones in vivo .
this may at first sight seem somewhat surprising , as the apparent direct contribution of mglu1 receptors to synaptic responses in thalamic slice experiments appears to be relatively small ( turner and salt , 2000 )
. this may in part be due to the relatively distal dendritic location of mglu1 receptors ( godwin et al .
, 1996 ; martin et al . , 1992 ; vidnyanszky et al . , 1996 ) , but it is also known that mglu1 receptor activation can have very non - linear effects on thalamic neuronal membrane properties ( mccormick and von krosigk , 1992 ; turner and salt , 2000 ; williams et al . , 1997 ) , and this would enhance responses mediated via ionotropic receptors ( e.g. nmda or ampa receptors ) . in addition direct positive modulation of nmda receptors by mglu receptors has been demonstrated , and we have previously shown that low , subthreshold , levels of mglu1 activation can enhance thalamic neurone responses nmda and ampa receptor activation ( salt and binns , 2000 ) . finally , it has been shown that activation of the mglu1-phospholipase c beta 4 ( plc beta 4 ) molecular pathway can enhance the tonic firing mode of thalamic nociceptive neurones ( cheong et al . , 2008 ) .
thus mglu1 receptors appear to be critically placed to play a dynamic role in controlling the responses to nociceptive stimuli of thalamic neurones . however , it is important to note that there is evidence to suggest some involvement of mglu5 receptors in thalamic nociceptive responses , although this is unlikely to arise from cortical input ( salt and binns , 2000 ) .
interestingly , both ro67 - 4853 and ly367385 appeared to have a much greater influence on thalamic nociceptive responses than the effects they produced on vibrissal responses reported by ourselves previously ( salt et al . , 2012 ) ; this is parallelled by the large effect of cortical inactivation on nociceptive responses that we have described compared to the lack of effect of cortical inactivation on vibrissal responses of ventrobasal thalamus neurones ( diamond et al . ,
our findings demonstrate the pivotal contribution that mglu1 receptors make to nociceptive processing at the thalamic level .
this is important in terms of understanding nociceptive mechanisms and in the design of novel therapeutics .
however , given that mglu1 receptors have been implicated in synaptic plasticity ( nicoletti et al . , 2011 ;
niswender and conn , 2010 ) , our findings suggest that plastic mechanisms may be activated under these conditions in vivo , and may underlie changes that may occur in chronic pain conditions .
moreover they underline the role that the cortical feedback may have in chronic pain and highlight the potential for development of pharmacological intervention targeting these mechanisms in the clinical treatment of chronic pain . | as postsynaptic metabotropic subtype 1 ( mglu1 ) receptors are present in the thalamus , we have investigated the effect of potentiating and antagonising mglu1 receptors on responses of thalamic neurones to noxious sensory stimulation .
extracellular recordings were made in vivo with multi - barrel iontophoretic electrodes from single neurones in the thalamus of urethane - anaesthetised rats .
responses to iontophoretic applications of the group i mglu agonist 3,5-dihydroxy - phenylglycine ( dhpg ) were selectively potentiated by co - application of the mglu1 positive allosteric modulator ro67 - 4853 , whereas they were selectively reduced upon co - application of the mglu1 receptor orthosteric antagonist ly367385 .
this indicates that thalamic dhpg responses are mediated primarily via mglu1 receptors , consistent with the high postsynaptic levels of this receptor in the thalamus .
furthermore , potentiation of dhpg responses by ro67 - 4853 were greater when the initial dhpg response was of a low magnitude .
ro67 - 4853 also potentiated responses of thalamic neurones to noxious thermal stimulation , whilst having little effect on the baseline activity of nociceptive neurones .
by contrast , nociceptive responses were reduced by ly367385 . in a further series of experiments we found that inactivation of somatosensory cortex by cooling resulted in a reduction of thalamic nociceptive responses .
these results underline the importance of mglu1 receptors in the processing of sensory information in the thalamus , particularly with respect to nociceptive responses .
furthermore , the involvement of mglu1 receptors may reflect the activity of descending cortico - thalamic afferents . |
background noise is pervasive in a wide range of our everyday environments including home , workplace , school , transportation , store , and recreational spaces . under noisy environments
, some people may experience difficulties understanding speech in noise whereas some may have less problems . considering the large variability in individual susceptibility to noise , nabelek , et al . quantified the lowest signal to noise ratio that a person could accept while listening to speech . this noise level that an individual can tolerate or accept
the anl test appears to be a quick , reliable , clinician - friendly procedure . unlike speech - in - noise recognition test , listener does not need to repeat the target speech they heard for anl measurement .
rather , the anl is calculated by difference between the most comfortable level for speech and the maximum level of background noise that the listener is willing to accept while following the target story .
although various types of sound soruces have been used as target sound such as reversed , unfamiliar discourses , and non - semantic sound sources , little research has focused the individual susceptibility to interference from background music . to our knowlege ,
only a few studies have reported anls to background music using different genres of music . in the previous study of nabelek , et al .
, ' light music ' of waiting room was first used as source of background noise to examine listeners ' anls to music . in gordon - hickey and moore , music of rock genre
was only used as background noise for anl measurement . comparing the results of young normal - hearing adults between two studies , nabelek and colleagues reported that ' light music ' was more demanding compared to multi - talker babble noise whereas gordon - hickey and moore found more acceptance of rock - genre music than babble noise . for the inconsistent finding
, the authors speculated a substantial impact of musical attributes such as music tempo , genre , familiarity , and musical experience . since no studies have examined the influence of music tempo and genre on anls , the present study aimed to determine whether music tempo and genre would affect individual anls .
in addition , this study investigated whether music familiarity and preference would influence the anls . to facilitate comparison with the previous finding of gordon - hickey and moore , this study grouped participants into low - anl group ( listeners with greater acceptance of noise ) and high - anl group ( listeners with lower acceptance of noise ) .
this would allow us to investigate whether music preference or familiarity would affect anls differently between low - anl and high - anl groups .
a total of forty - one ( 21 males , 20 females ) native korean listeners participated in this study .
accoridng to the previous criteria , the participants consisted of 29 low - anl listeners ( 6 db ) and 12 high - anl listeners ( 14 db ) .
the mean age of low - anl group ( n=29 ) was 47.2 years of age [ standard deviation ( sd ) : 23.4 ] , and the mean age of high - anl group ( n=12 ) was 47.1 years of age ( sd : 17.0 ) .
the mean puretone threshold of two groups was less than 20 db hl from 250 to 2000 hz , and less than 30 db hl at 4000 and 6000 hz .
analyses of independent t - test showed that the ages and the octave - scale puretone thresholds from 250 to 6000 hz did not significantly differ ( p>0.05 for all frequencies ) between low - anl and high - anl groups .
additional analyses of pearson correlation verified no significant relations between anls and puretone threshold average and also between anls and ages of individuals , consistent with previous finding . before the experimental test
, participants were informed of anticipated risk and benefit and all were agreed to participation in research before the experimental testing . for the anl measure ,
a recorded discourse of a male talker in korean was used as target story . as background noise , anls
were tested in six different music categories consisting of three music genres ( korean - pop music : k - pop , popular music : pop , and classical music : classical ) and two types of music tempo ( fast and slow ) .
each of the six categories included three music samples , resulting in 18 music samples as total , as displayed in table 1 . selecting as either fast or slow tempo
first , as an objective standard , the number of beats per minute ( bpm ) was considered , as in the previous criteria .
thus , 94 or more bpm was required to be fast tempo music whereas 72 or less bpm for the slow tempo music . especially for the classical music ,
the tempo was based on the quarter - notes per minute ( qpm ) such that adagio ( 60 - 80 qpm ) classical music was chosen as slow classical music and allegro ( 120 - 160 qpm ) was selected as fast classical music .
second , as described in the previous studies which measured anls to background music , a single instrumental part was avoided such that vocal parts with lyrics were selected as music samples of k - pop and pop genres . in contrast , the instrumental part without lyrics was selected as music sample of classical genre .
the average root mean square value of each wavefile was equated via adobe audition version 3.0 ( adobe systems incorporated , san jose , ca , usa ) with 16-bit resolution and a sampling rate of 44.1 khz .
all the stimuli were delivered via clinical audiometer ( madsen obiter 922 , gn otometrics , taastrup , denmark ) connected to an external cd player ( dej017 , sony , tokyo , japan ) and a loudspeaker at 0 degrees azimuth to the subject .
prior to the experimental test , a practice with verbal and written instructions in korean was given to ensure whether the participant understood task correctly .
subjects ' anls were obtained based on the procedure of the previous study which first introduced anl . for the anl measure ,
each participant was required to adjust level of a target story in 2 db steps to find individual 's most comfortable level ( mcl ) .
while listening to and following the target message at mcl , the listener was then required to adjust level of background music in 2 db steps to find the maximum level that the listener was willing to accept , called background noise level ( bnl ) .
the anl was determined by subtracting bnl from mcl ( anl = mcl - bnl ) .
after the anl test , participants were asked to judge the degree of familiarity and preference on each music sample using a 4-point scales , as used in the previous study on music familiarity . on this questionnaire , the greater numbers correspond to the higher levels of familiarity or preference . to complete all the task ,
first , a 2 ( group)2 ( tempo)3 ( genre ) three - way mixed - model analysis of variance ( anova ) was conducted on anls as dependent variable .
group was a between - subjects variable ( low - anl and high - anl ) , and music tempo ( fast and slow ) and music genre ( k - pop , pop , and classical ) were used as two within - subjects variables .
second , in order to examine any relationship of anls with subjective responses , non - parametric analyses of spearman 's rank - order correlation were conducted on music preference and music familiarity for low - anl and high - al groups , separately . for analyses with a lack of sphericity , the degrees of freedom and f values were adjusted with the greenhouse - geisser correction . if necessary , any post - hoc multiple comparisons were conducted .
a total of forty - one ( 21 males , 20 females ) native korean listeners participated in this study .
accoridng to the previous criteria , the participants consisted of 29 low - anl listeners ( 6 db ) and 12 high - anl listeners ( 14 db ) .
the mean age of low - anl group ( n=29 ) was 47.2 years of age [ standard deviation ( sd ) : 23.4 ] , and the mean age of high - anl group ( n=12 ) was 47.1 years of age ( sd : 17.0 ) .
the mean puretone threshold of two groups was less than 20 db hl from 250 to 2000 hz , and less than 30 db hl at 4000 and 6000 hz .
analyses of independent t - test showed that the ages and the octave - scale puretone thresholds from 250 to 6000 hz did not significantly differ ( p>0.05 for all frequencies ) between low - anl and high - anl groups .
additional analyses of pearson correlation verified no significant relations between anls and puretone threshold average and also between anls and ages of individuals , consistent with previous finding . before the experimental test
, participants were informed of anticipated risk and benefit and all were agreed to participation in research before the experimental testing .
for the anl measure , a recorded discourse of a male talker in korean was used as target story . as background noise , anls
were tested in six different music categories consisting of three music genres ( korean - pop music : k - pop , popular music : pop , and classical music : classical ) and two types of music tempo ( fast and slow ) .
each of the six categories included three music samples , resulting in 18 music samples as total , as displayed in table 1 . selecting as either fast or slow tempo
first , as an objective standard , the number of beats per minute ( bpm ) was considered , as in the previous criteria .
thus , 94 or more bpm was required to be fast tempo music whereas 72 or less bpm for the slow tempo music . especially for the classical music ,
the tempo was based on the quarter - notes per minute ( qpm ) such that adagio ( 60 - 80 qpm ) classical music was chosen as slow classical music and allegro ( 120 - 160 qpm ) was selected as fast classical music .
second , as described in the previous studies which measured anls to background music , a single instrumental part was avoided such that vocal parts with lyrics were selected as music samples of k - pop and pop genres .
in contrast , the instrumental part without lyrics was selected as music sample of classical genre .
the average root mean square value of each wavefile was equated via adobe audition version 3.0 ( adobe systems incorporated , san jose , ca , usa ) with 16-bit resolution and a sampling rate of 44.1 khz .
all the stimuli were delivered via clinical audiometer ( madsen obiter 922 , gn otometrics , taastrup , denmark ) connected to an external cd player ( dej017 , sony , tokyo , japan ) and a loudspeaker at 0 degrees azimuth to the subject .
prior to the experimental test , a practice with verbal and written instructions in korean was given to ensure whether the participant understood task correctly .
subjects ' anls were obtained based on the procedure of the previous study which first introduced anl . for the anl measure ,
each participant was required to adjust level of a target story in 2 db steps to find individual 's most comfortable level ( mcl ) .
while listening to and following the target message at mcl , the listener was then required to adjust level of background music in 2 db steps to find the maximum level that the listener was willing to accept , called background noise level ( bnl ) .
the anl was determined by subtracting bnl from mcl ( anl = mcl - bnl ) .
after the anl test , participants were asked to judge the degree of familiarity and preference on each music sample using a 4-point scales , as used in the previous study on music familiarity . on this questionnaire
, the greater numbers correspond to the higher levels of familiarity or preference . to complete all the task ,
all the statistical analyses were performed using spss version 20.0 ( ibm corp . , armonk
first , a 2 ( group)2 ( tempo)3 ( genre ) three - way mixed - model analysis of variance ( anova ) was conducted on anls as dependent variable .
group was a between - subjects variable ( low - anl and high - anl ) , and music tempo ( fast and slow ) and music genre ( k - pop , pop , and classical ) were used as two within - subjects variables .
second , in order to examine any relationship of anls with subjective responses , non - parametric analyses of spearman 's rank - order correlation were conducted on music preference and music familiarity for low - anl and high - al groups , separately . for analyses with a lack of sphericity , the degrees of freedom and f values were adjusted with the greenhouse - geisser correction . if necessary , any post - hoc multiple comparisons were conducted .
table 2 shows the mean anl and standard deviation for low - anl and high - anl groups when background music differed by music tempo and genre . when the background music of slow tempo was presented , the mean anls ranged from 2.2 to 2.5 db for low - anl listeners and ranged from 17.3 to 17.8 db for high - anl listeners across three music genres . when the fast - tempo music was presented , the mean anls of low - anl group were 4.1 , 3.0 , and 3.1 db and the mean anls of high - anl group were 17.5 , 16.1 , and 16.1 db for k - pop , pop , and classical music , respectively .
a 223 three - way mixed - model anova was performed on anls and the results showed that the main effect of group was significant [ f(1 , 39)=281.5 ] . also , we found that music genre significantly influenced the anls [ f(1.73 , 67.64)=5.36 ] but music tempo did not significantly influence the anls .
results from bonferroni - adjusted multiple paired - comparisons revealed that the anls to k - pop music were significantly greater than the anls to pop or classical music .
a two - way interaction between tempo and group was significant [ f(1 , 39)=9.78 ] , showing higher anls with fast music for low - anl group yet in opposite for high - anl groups .
other two - way interactions as well as a three - way interaction among tempo , genre , and group were not significiant .
this indicates that the k - pop music which includes vocal part with korean lyrics yields the greatest amount of interference as background noise .
this study investigated individual music familiarity and preference on each music sample in order to examine whether the anls would be related to individual 's familiarity and preference .
the mean values of music familiarity was 2.5 ( sd : 0.6 ) and 2.4 ( sd : 0.8 ) for low - anl and high - anl groups , respectively .
the mean values of music preference was 2.3 ( sd : 0.5 ) and 2.1 ( sd : 0.7 ) for low - anl and high - anl groups , respectively .
the independent t - test showed that the subjective responses did not statistically differ between low - anl and high - anl goups on both music familiarity [ t(39)=0.37 ] and preference [ t(39)=1.01 ] .
non - parametric analyses of spearman 's rank - order correlation were administered to determine any association between individual noise acceptance and their subjective preference or familiarity to music samples . in order to investigate this , we examined relationship between individual anls and subjective responses for low - anl and high - anl groups .
table 3 shows the spearman 's coefficients rho for low - anl and high - anl groups .
as displayed , for low - anl individuals , their acceptance of background music was relatively unaffected by music familiarity and preference , regardless of music genre .
however , the results on correlated measures revealed a different tendency in high - anl group . for high - anl individuals ,
the anls were significantly and negatively related to subjective ratings of music familiarity and preference when the music samples were from k - pop ( -0.83<rs<-0.73 ) or pop genre ( -0.87<rs<-0.75 ) .
that is , the subjective preference or familiarity of background noise would affect noise acceptance only for the high - anl listeners , rather than for all listeners .
table 2 shows the mean anl and standard deviation for low - anl and high - anl groups when background music differed by music tempo and genre . when the background music of slow tempo was presented , the mean anls ranged from 2.2 to 2.5 db for low - anl listeners and ranged from 17.3 to 17.8 db for high - anl listeners across three music genres . when the fast - tempo music was presented , the mean anls of low - anl group were 4.1 , 3.0 , and 3.1 db and the mean anls of high - anl group were 17.5 , 16.1 , and 16.1 db for k - pop , pop , and classical music , respectively .
a 223 three - way mixed - model anova was performed on anls and the results showed that the main effect of group was significant [ f(1 , 39)=281.5 ] . also , we found that music genre significantly influenced the anls [ f(1.73 , 67.64)=5.36 ] but music tempo did not significantly influence the anls .
results from bonferroni - adjusted multiple paired - comparisons revealed that the anls to k - pop music were significantly greater than the anls to pop or classical music .
a two - way interaction between tempo and group was significant [ f(1 , 39)=9.78 ] , showing higher anls with fast music for low - anl group yet in opposite for high - anl groups .
other two - way interactions as well as a three - way interaction among tempo , genre , and group were not significiant .
this indicates that the k - pop music which includes vocal part with korean lyrics yields the greatest amount of interference as background noise .
this study investigated individual music familiarity and preference on each music sample in order to examine whether the anls would be related to individual 's familiarity and preference .
the mean values of music familiarity was 2.5 ( sd : 0.6 ) and 2.4 ( sd : 0.8 ) for low - anl and high - anl groups , respectively .
the mean values of music preference was 2.3 ( sd : 0.5 ) and 2.1 ( sd : 0.7 ) for low - anl and high - anl groups , respectively .
the independent t - test showed that the subjective responses did not statistically differ between low - anl and high - anl goups on both music familiarity [ t(39)=0.37 ] and preference [ t(39)=1.01 ] .
non - parametric analyses of spearman 's rank - order correlation were administered to determine any association between individual noise acceptance and their subjective preference or familiarity to music samples . in order to investigate this , we examined relationship between individual anls and subjective responses for low - anl and high - anl groups .
table 3 shows the spearman 's coefficients rho for low - anl and high - anl groups .
as displayed , for low - anl individuals , their acceptance of background music was relatively unaffected by music familiarity and preference , regardless of music genre .
however , the results on correlated measures revealed a different tendency in high - anl group . for high - anl individuals ,
the anls were significantly and negatively related to subjective ratings of music familiarity and preference when the music samples were from k - pop ( -0.83<rs<-0.73 ) or pop genre ( -0.87<rs<-0.75 ) .
that is , the subjective preference or familiarity of background noise would affect noise acceptance only for the high - anl listeners , rather than for all listeners .
although various types of target stories and listening conditions have been used for anl measure , a source of background sound has been limited other than use of multitalker babble noise . despite a widespread and common use of music in everyday life ,
gordon - hickey and moore who used one genre of rock as music samples suggested a need of more research to explore individual susceptibility to background music with controlling musical attributes such as tempo , genre , and instrument composition . this has driven this study to investigate the effect of music tempo and genre on individual anls to music .
results of this study showed that k - pop music was relatively less accepted as background noise , resulting in a relatively greater amont of noise distraction ( higher anl ) compared to pop or classical music .
interestingly , the significant effect of genre on anls was similar in both low - anl and high - anl groups .
as one of the possible reasons , the native korean participants could have been greatly annoyed by k - pop music due to meaningful content of k - pop songs .
this seems consistent with previous anl measures which compared anls across five types of competing speech maskers ( e.g. , 1-male , 1-female , 2-talker , 4-talker , and 8-talker speech masker ) . considering the features of relatively reduced temporal fluctuations and unintelligible meaning in babble noise as background
, researchers have examined individuals ' noise acceptance as a function of the number of the competing talkers .
the findings were congruent such that acceptance of background noise was significantly poorer ( or higher anl ) when the background speech masker became more meaningful ( i.e. , 1- or 2-talker noise ) for young normal - hearing listeners , adult hearing aid users , and adults who use cochlear implant .
thus , selecting a type of the noise source should be carefully considered for clinical measrues of anls and also when comparing data across anl studies who used different materials .
although it was not the primary interest of this study , no relation was found between anls and individuals ' age or hearing threshold , consistent with previous findings .
this supports the previous findings that anl might be an inherent characteristic of individual , which is unchangeable with age or acquired hearing loss of listeners .
recent research revealed that the anls could be based on subjective loudness , self - control level , and personality .
considering the substantial impact of subjective and inherent characteristics of the individuals on their anls , it seems important to investigate whether the greater annoyance from k - pop music would be associated with self - rating responses on music familiarity and preference . to clear this out , the present study explored any association between anls and individual familiarity or preference to music samples .
the results revealed a different tendency on correlated measures between listeners with low - anl ( n=29 ) and high - anl ( n=12 ) .
results showed that the anls of low - anl group were independent of their subjective responses of music familiarity and preference .
this seems similar with previous finding who reported no significant correlation between anls and individual preference to background noise .
gordon - hickey and moore also showed no association between anls and participants ' music preference .
however , we found that the anls of high - anl group depended on music familiarity and preference .
expected result to observe was that the listeners would accept more noise ( lower anl ) with less familiar and less preferred background music , meaning a positive association between anls and subjective responses ( music familiarity and preference ) .
unexpectedly , negative correlations were observed between anls and subjective responses of high - anl group ( less acceptance of background music for less familiar or less preferred music ) .
possibly , the relatively less familiar or less preferred music could introduce new conflicting cues , resulting in more distraction when following a target story .
if a listener had a good strategy to suppress familiar music entirely , then the familiarity of music would be beneficial , as reported using word identification .
however , a limitation of the study is the relatively small sample size especially for high - anl group ( n=12 ) such that more research is needed to determine whether subjective familiarity or preference on noise can affect a person 's capacity to accept background noise .
the purpose of the current study was to evaluate the effects of music genre and tempo on the anls to background music .
results showed that , overall , the anls did not significantly differ by music tempo .
the anls depended on the genre of background music . since k - pop music with more meaningful lyrics
was poorly accepted than pop or classical music for both low - anl and high - anl groups , the degree of informational masking would decrease acceptance of background noise .
the subjective familiarity and preference of noise had a great impact on anls only for high - anl group . | background and objectivesacceptable noise level ( anl ) is a measure of the maximum background noise level ( bnl ) that a person is willing to tolerate while following a target story .
although researchers have used various sources of target sound in anl measures , a limited type of background noise has been used . extending the previous study of gordon - hickey & moore ( 2007 ) , the current study determined the effect of music genre and tempo on anls as possible factors affecting anls .
we also investigated the relationships between individual anls and the familiarity of music samples and between music anls and subjective preference.subjects and methodsforty - one participants were seperated into two groups according to their anls , 29 low - anl listeners and 12 high - anl listeners .
using korean anl material , the individual anls were measured based on the listeners ' most comfortable listening level and bnl .
the anls were measured in six conditions , with different music tempo ( fast , slow ) and genre ( k - pop , pop , classical ) in a counterbalanced order.resultsoverall , anls did not differ by the tempo of background music , but music genre significantly affected individual anls .
we observed relatively higher anls with k - pop music and relatively lower anls with classical music .
this tendency was similar in both low - anl and high - anl groups .
however , the subjective ratings of music familiarity and preference affected anls differently for low - anl and high - anl groups .
in contrast to the low - anl listeners , the anls of the high - anl listeners were significantly affected by music familiarity and preference.conclusionsthe genre of background music affected anls obtained using background music .
the degree of music familiarity and preference appears to be associated with individual susceptibility to background music only for listeners who are greatly annoyed by background noise ( high - anl listeners ) . |
main tumour sites are the sacrum ( 5060% ) , the skull base ( 2535% ) and the spine , mainly cervical ( 15% ) .
lung , bone , liver or soft tissue metastases sometimes arise , but local recurrence is the main cause of death [ 1 , 2 ] .
initial treatment is based on complete surgical resection . however , safe margins are difficult to achieve due to the vicinity of vital or functional anatomic structures
. radiation therapy is often performed after surgery to limit the risk of a local recurrence .
proton beam therapy is the most recommended technique in order to deliver a high dose to the tumour with limited damage to adjacent tissues .
conventional cytotoxic drugs are not active against this tumour type . a modest but significant activity of imatinib , an inhibitor of platelet - derived growth factor receptor ( pdgfr ) and c - kit , has been described in several case reports and in a phase ii study [ 4 , 5 , 6 ] . more recently ,
clinical activity of erlotinib , a tyrosine kinase inhibitor of the epidermal growth factor receptor ( egfr ) , was observed [ 7 , 8 , 9 , 10 ] .
we report a new , well - documented case of a locally advanced chordoma that responded to erlotinib after the failure of imatinib .
an mri showed a heterogeneous mass involving the sphenoid bone , with suprasellar and left cavernous sinus extension .
histological analysis demonstrated the presence of large cells disposed in heaps with round nuclei leading to the diagnosis of clivus chordoma .
a second partial surgical resection was performed in january 2006 due to tumour progression into the upward sella and close contact to the chiasma .
diplopia recurred in october 2009 due to paralysis of the right abducens nerve ( vi ) . in september 2011 ,
ptosis of the right palpebra developed , and it was accompanied by a deficit of the right oculomotor nerve ( iii ) and by mydriasis .
imatinib ( 400 mg / day , then 800 mg / day ) was given for 5 months , but the tumour nevertheless progressed toward the frontal lobe ( fig .
to date ( july 2014 ) , ptosis persists , but without any new neurological symptoms .
the tolerance of erlotinib was relatively good , with a moderate rash on the face and diarrhea that was controlled by loperamide .
activation of pdgfra and pdgfrb was documented in chordoma [ 11 , 12 ] , leading to the clinical evaluation of imatinib , a pdgf inhibitor [ 4 , 5 ] .
a phase ii study has been completed using 800 mg / day imatinib . among 50 patients ,
egfr expression and activation is also involved in chordoma progression . in the first study investigating egfr expression in chordoma ,
another study reported an egfr expression in 83% of primary chordomas and 97% of recurrent chordomas among 52 patients , while a third study showed that the egfr was expressed in 60% of chordomas and activated in 50% of cases among 173 chordomas .
egfr is then the most significantly activated receptor tyrosine kinase in chordomas , and therefore a potential therapeutic target . in vitro studies
have shown that pharmacological inhibition of egfr had an anti - tumour effect on the u - ch1 human chordoma cell line .
tyrphosin ( ag1478 ) , an egfr inhibitor , diminished cell proliferation and egfr phosphorylation .
erlotinib limits egfr phosphorylation by binding to the intracellular part of the egfr tyrosine kinase domain .
downstream inhibition of the ras / mek pathway inhibits the proliferation and survival of sensitive cancer cells .
activation of egfr by egf enhanced cell proliferation in vitro , but erlotinib decreased proliferation of the u - ch1 and c24 human chordoma cell lines in a dose - dependent fashion [ 16 , 17 ] . in the same study , treatment of a patient - derived xenograft with
erlotinib resulted in a significant reduction of tumour volume compared to control , and molecular analyses revealed reduced phosphorylation of the tyr845 residue of egfr .
this molecular rationale led to a compassionate use of erlotinib in patients with relapsed chordoma , especially when the disease progressed despite imatinib treatment . to date , there are 4 case reports describing the activity of erlotinib when used in association with bevacizumab in chordoma [ 9 , 10 ] .
the role that can be ascribed to erlotinib is then difficult to define , but to our knowledge , bevacizumab alone has no effect on chordoma .
only 2 case reports have described a tumour response with erlotinib treatment alone . in the first case ,
a 30% tumour reduction was observed with erlotinib treatment in a sacral metastatic chordoma with gluteal mass and iliac lymph nodes that was resistant to imatinib .
a 70% regression of a thoracic chordoma with lung metastases and cervical lymph nodes were registered after 7 months of erlotinib treatment , while the disease was refractory to imatinib .
our study is the 7th study to be published to date investigating the role of erlotinib in chordoma , and the third using erlotinib alone . here
, we report the longest response duration to erlotinib described in the literature to date ( 28 + months ) .
our case report corroborates previous studies , showing that erlotinib is an effective drug in patients with advanced chordoma , even those refractory to imatinib . a prospective clinical trial comparing erlotinib and imatinib as a first - line treatment of recurrent chordoma is mandatory .
| chordoma is a rare malignant axial tumour that develops from embryonic remnants of the notochord .
surgery and irradiation are the standard initial treatment .
however , local recurrence is frequent and cytotoxic chemotherapy is inefficient .
transient activity of imatinib , a platelet - derived growth factor receptor inhibitor , was described in a phase ii study .
activity of epidermal growth factor receptor ( egfr ) inhibitors ( erlotinib , gefitinib ) has also been shown in a few recent case reports .
we describe a 68-year - old female in whom clivus chordoma recurred after surgery and radiotherapy .
the tumour progressed despite imatinib treatment . a partial and sustained response
( 28 + months ) was obtained using erlotinib , an egfr inhibitor .
erlotinib should be evaluated in a prospective trial investigating new potential therapies against recurrent chordoma . |
combined hyperlipidemia ( chl ) , a highly atherogenic lipid disorder characterized by increased low - density lipoprotein cholesterol ( ldl - c ) , elevated triglycerides ( tgs ) and decreased high - density lipoprotein cholesterol ( hdl - c ) is common in patients with type 2 diabetes mellitus . moreover
, metabolic abnormalities such as predominance of small dense ldl particles and increased glycation of ldl raise the atherogenic risk in these patients .
cardiovascular disease or coronary artery disease is the major cause of death in both men and women in the developed countries .
statins have been shown to reduce atherosclerosis related morbidity and mortality in patients with diabetes mellitus and fibrates can decrease tg concentrations and elevate hdl - c , thus reducing the cardiovascular morbidity and mortality .
recent studies showed that statin or fibrate monotherapy can improve the lipid profile in patients with type 2 diabetes mellitus .
however , these affect different aspects of lipoprotein metabolism . hence , it is difficult to modify the lipid profile of patients with type 2 diabetes mellitus using the monotherapy with either a statin or a fibrate according to the recent investigations of the american diabetes association .
hypolipidemic effect of atorvastatin is due to inhibition of hydroxymethylglutaryl - coa reductase and decrease in ldl - c is also due to up regulation of ldl receptor activity .
outcome trials of statins have proved conclusively that these classes of drugs decrease ldl - c levels , resulting in a significant reduction of cardiovascular events in many high - risk patients .
it has been shown that statins significantly reduced circulating levels of all major ldl subspecies ; light , intermediate and dense .
effects such as vasodilatation , plaque stabilization , antioxidant , anti - inflammatory and antithrombotic effects .
fibrates , commonly referred to as peroxisome proliferator activated receptor- ( ppar- ) agonists , are subfamily of the nuclear receptor superfamily that have been noted to be naturally activated by ligands such as free fatty acids and eicosanoids .
they significantly decrease tg levels , ldl - c and increase hdl - c and hence help in reducing the cardiovascular events .
fibrates decrease the production of tg - rich lipoproteins and increase the catabolism of tgs by the inducing lipoprotein lipase and also reduces expression of apolipoprotein c - iii through activation of ppar-. an effective therapeutic approach for chl is a combination of statin and fibrate .
studies comparing lipid lowering therapies have shown that atorvastatin reduces all ldl sub fractions , whereas fenofibrate reduces ldl density .
this is due to a more pronounced reduction of the densest ldl fraction ( ldl-6 ) by fenofibrate compared with atorvastatin .
the literature on statin - fibrate combinations in patients with type 2 diabetes and chl is limited .
hence , an open label , prospective , randomized , parallel study was undertaken to investigate the hypothesis that combination therapy with atorvastatin and fenofibrate will be more effective than monotherapy with a statin / fibrate to prevent coronary heart disease in patients with type 2 diabetes mellitus .
an open - labeled , randomized , prospective study was conducted from march 2009 to june 2010 in patients of type 2 diabetes mellitus associated with hyperlipidemia , attending the out - patient department of endocrinology , sri venkateswara institute of medical sciences , tirupati .
the study was conducted by the department of pharmacology , sri venkateswara medical college , tirupati .
patients of either gender in the age group of 30 - 70 years diagnosed as type 2 diabetes mellitus with hyperlipidemia ( total cholesterol [ tc ] > 200 mg / dl , tgs > 150 mg / dl , ldl - c > 100 mg / dl , hdl - c < 40 mg/ dl ) based on criteria laid down by national cholesterol education program ( ncep adult treatment panel iii ) guidelines were included in the study .
patients on concurrent therapy with beta blockers , thiazides , oral contraceptives pills , and cyclosporine , erythromycin and azole antifungals were excluded .
patients having liver dysfunction ( levels of transaminases 1.5 or more times the upper limit of normal , renal dysfunction ( serum creatinine greater than 1.6 mg / dl ) and pregnant and lactating women were also excluded from the study .
the institutional ethical committee approved the study protocol and informed consent was obtained from all patients before enrollment after detailed explanation of possible adverse effects of the drug combinations .
patients were on the national cholesterol expert panel step 1 hypolipedemic diet , which limits dietary intake of cholesterol to 300 mg / day , saturated fats 10% of total energy intake and total fats to 30% total energy intake during the study .
patients who fulfilled the inclusion / exclusion criteria were randomly divided into two groups of 30 each by simple randomization using computer generated random numbers .
all laboratory investigations were obtained after the patients had fasted for 12 h overnight and included the estimation of serum lipid parameters ( tc , hdl - c , ldl - c , tgs and vldl - c ) as well as fasting blood sugar ( fbs ) , glycated hemoglobin ( hba1c ) , hb% , renal function tests and liver function tests were measured during screening in the same laboratory .
patient demographic and baseline characteristics group 1 received atorvastatin 10 mg / day and group 2 received combination of atorvastatin 10 mg / day and fenofibrate 145 mg / day for 12 weeks orally at night .
patients were assessed after 12 weeks and were asked to report immediately if they developed unusual muscle soreness or pain throughout the study .
lipid profile , fbs was done after 12 weeks . in case of history of muscle pain ,
serum cholesterol and tgs were determined using an enzymatic colorimetric assay and hdl - c was determined by phosphotungstic acid method .
ldl - c was calculated using friedewald 's formula ( ldl = tc - [ tg/5 + hdl ] ) .
sample size was calculated taking into consideration the mean values and standard deviation from the study done by athyro et al .
the data obtained were analyzed using descriptive statistics and paired and unpaired student t - test to compare results within the group and between groups .
an open - labeled , randomized , prospective study was conducted from march 2009 to june 2010 in patients of type 2 diabetes mellitus associated with hyperlipidemia , attending the out - patient department of endocrinology , sri venkateswara institute of medical sciences , tirupati .
the study was conducted by the department of pharmacology , sri venkateswara medical college , tirupati .
patients of either gender in the age group of 30 - 70 years diagnosed as type 2 diabetes mellitus with hyperlipidemia ( total cholesterol [ tc ] > 200 mg / dl , tgs > 150 mg / dl , ldl - c > 100 mg / dl , hdl - c < 40 mg/ dl ) based on criteria laid down by national cholesterol education program ( ncep adult treatment panel iii ) guidelines were included in the study .
patients on concurrent therapy with beta blockers , thiazides , oral contraceptives pills , and cyclosporine , erythromycin and azole antifungals were excluded .
patients having liver dysfunction ( levels of transaminases 1.5 or more times the upper limit of normal , renal dysfunction ( serum creatinine greater than 1.6 mg / dl ) and pregnant and lactating women were also excluded from the study .
the institutional ethical committee approved the study protocol and informed consent was obtained from all patients before enrollment after detailed explanation of possible adverse effects of the drug combinations .
patients were on the national cholesterol expert panel step 1 hypolipedemic diet , which limits dietary intake of cholesterol to 300 mg / day , saturated fats 10% of total energy intake and total fats to 30% total energy intake during the study .
patients who fulfilled the inclusion / exclusion criteria were randomly divided into two groups of 30 each by simple randomization using computer generated random numbers .
all laboratory investigations were obtained after the patients had fasted for 12 h overnight and included the estimation of serum lipid parameters ( tc , hdl - c , ldl - c , tgs and vldl - c ) as well as fasting blood sugar ( fbs ) , glycated hemoglobin ( hba1c ) , hb% , renal function tests and liver function tests were measured during screening in the same laboratory .
group 1 received atorvastatin 10 mg / day and group 2 received combination of atorvastatin 10 mg / day and fenofibrate 145 mg / day for 12 weeks orally at night .
patients were assessed after 12 weeks and were asked to report immediately if they developed unusual muscle soreness or pain throughout the study .
lipid profile , fbs was done after 12 weeks . in case of history of muscle pain ,
serum cholesterol and tgs were determined using an enzymatic colorimetric assay and hdl - c was determined by phosphotungstic acid method .
ldl - c was calculated using friedewald 's formula ( ldl = tc - [ tg/5 + hdl ] ) .
sample size was calculated taking into consideration the mean values and standard deviation from the study done by athyro et al .
the data obtained were analyzed using descriptive statistics and paired and unpaired student t - test to compare results within the group and between groups .
from the initial 30 patients in group 2 , one patient terminated the study for personal reasons and one patient interrupted the study because of mild muscle pain .
they received atorvastatin ( n = 30 ) and combination of atorvastatin and fenofibrate ( n = 28 ) for a period of 3 months .
there was a significant reduction ( p < 0.001 ) in tc ( 28% ) , tgs ( 20% ) , ldl - c ( 37% ) , vldl - c ( 20% ) and insignificant increase in hdl - c in atorvastatin group at the end of 3 months as compared to pretreatment values .
there was a significant reduction ( p < 0.001 ) in tc ( 31% ) , tgs ( 39% ) , ldl - c ( 33% ) , vldl - c ( 40% ) and insignificant decrease in hdl - c in combination treatment group at the end of 3 months as compared with pretreatment values as represented in table 2 .
comparison of changes in the lipid profile between the two treatment groups after 12 weeks the reduction in tc , tgs , vldl - c in combination treatment group was statistically significant ( p < 0.001 ) when compared to atorvastatin group alone .
the observed difference in a decrease in ldl - c and change in hdl - c was not statistically significant between the two treatment groups [ table 2 ] .
there was a significant reduction ( p < 0.001 ) in tc ( 28% ) , tgs ( 20% ) , ldl - c ( 37% ) , vldl - c ( 20% ) and insignificant increase in hdl - c in atorvastatin group at the end of 3 months as compared to pretreatment values .
there was a significant reduction ( p < 0.001 ) in tc ( 31% ) , tgs ( 39% ) , ldl - c ( 33% ) , vldl - c ( 40% ) and insignificant decrease in hdl - c in combination treatment group at the end of 3 months as compared with pretreatment values as represented in table 2 .
comparison of changes in the lipid profile between the two treatment groups after 12 weeks the reduction in tc , tgs , vldl - c in combination treatment group was statistically significant ( p < 0.001 ) when compared to atorvastatin group alone .
the observed difference in a decrease in ldl - c and change in hdl - c was not statistically significant between the two treatment groups [ table 2 ] .
type 2 diabetes mellitus is one of the most common chronic disease and is associated with co - morbidities such as obesity , hypertension , hyperlipidemia and cardiovascular disease , which together , constitute metabolic syndrome .
there is evidence to suggest that diabetes is more common in females than males . in recent years
an increase in number of diabetic males resulted in an equal prevalence rates for both males and females in some societies . in our study , we have analyzed the effect of atorvastatin 10 mg once daily and combination of atorvastatin 10 mg and fenofibrate 145 mg once daily in type 2 diabetes mellitus patients associated with hyperlipidemia .
both treatments had decreased tc , tg , ldl - c , vldl - c , but the reduction of tc , tg , vldl - c was more and statistically significant in combination treatment group when compared with atorvastatin group alone at the end of 12 weeks .
the observed difference in decrease in ldl - c and change in hdl - c was not statistically significant between the two treatment groups .
a similar study , which compared the effect of atorvastatin ( 20 mg / day ) alone with micronized fenofibrate ( 200 mg / day ) monotherapy and in combination with fenofibrate in type 2 diabetes mellitus patients with chl where in the patients were in the age group of 44 - 69 years .
the combination treatment had reduced tc by 37% , ldl - c by 46% , tgs by 50% and increased hdl - c by 46% ( p < 0.0001 for all ) and these changes were significantly better than those of monotherapy .
no significant adverse events were recorded during the study . in another study , combination of simvastatin ( 20 mg / day)and bezafi brate ( 400 mg / day )
had reduced tc by 23% and ldl - c by 29% but had significantly reduced tg levels by 42% and increased hdl - c by 25% . of the 148 patients from this study , 2 had presented with myopathy . in action to control cardiovascular risk in diabetes lipid trial it was found that combination therapy with the use of fenofibrate and simvastatin ( at a daily dose of 40 mg or less ) did not reduce rates of cardiovascular disease as compared with simvastatin alone .
the primary risk of using statins in combination with fibrates is believed to be hepatotoxicity and myopathy . in most studies
however , due to limited number of patients and the short term follow - up , we can not draw any definite conclusions .
hence , further studies in a large number of patients and for longer duration are necessary to assess the long - term safety of this combination . | mixed dyslipidemia is characterized by increased low - density lipoprotein cholesterol ( ldl - c ) elevated triglycerides ( tgs ) and decrease high - density lipoprotein cholesterol ( hdl - c ) .
it is more common in diabetes and is associated with an increased risk of coronary artery disease .
monotherapy with statins or fibrates may not effectively control all lipid parameters .
the atorvastatin - fenofibrate combination has been shown to have highly beneficial effect on lipid parameters in type 2 diabetes associated with combined hyperlipidemia ( chl ) . in an open - label study
, we evaluated the efficacy of atorvastatin alone and in combination with fenofibrate in 60 types 2 diabetes mellitus patients associated with hyperlipidemia . patients were randomly assigned to receive atorvastatin 10 mg ( group 1 ) or combination of atorvastatin 10 mg and fenofibrate 145 mg ( group 2 ) once daily for 12 weeks .
the effect of drugs on lipid profile was evaluated before and after treatment .
after 12 weeks , the reduction in total cholesterol ( tc ) , tgs , ldl - c , vldl - c was 28% , 20% , 37% and 20% in group 1 ( p < 0.001 for all ) as compared with 31% , 39% , 33% and 40% in group 2 ( p < 0.001 for all ) .
there was insignificant rise in hdl - c in group 1 ( p = 0.71 ) and insignificant decrease in hdl - c ( p = 0.70 ) in group 2 . during the combination therapy ,
the decrease in tc , tgs and vldl - c was greater than atorvastatin alone . the combination of atorvastatin with fenofibrate in type 2 diabetes patients with chl may have a favorable effect on some major coronary artery disease risk factors . |
as characterized by hanchanale and coworkers , the perception of botulinum toxin a has been transformed from poison to a healing agent .
information concerning botulinum neurotoxin serotypes , molecular structures , substrate specificities , mechanisms of zinc - dependent peptide hydrolysis , ion channel formation , and other detailed topics has been extensively reviewed [ 24 ] .
of the seven immunologically distinct serotypes ( a f ) from clostridium botulinum and several other species , type a is the best characterized and is among the most potent of all toxins .
the neurotoxin is initially expressed as a single polypeptide of nearly 1300 amino acid residues ( mw ~150 kda ) .
crude toxin extracts ( mw ranges from ~300 to 900 kda ) contain several nontoxic ancillary proteins that form a complex with the neurotoxin .
when ingested , these additional proteins are thought to protect the neurotoxin against austere environments such as those found in the certain regions of the gastrointestinal tract .
the neurotoxin is posttranslationally modified to form two chains that are covalently bridged with a disulfide bond .
the light ( l ) chain ( mw ~50 kda ) has zinc - dependent proteolytic activity , while the heavy ( h ) chain contains the translocation and binding domains .
subsequent to binding to specific receptors ( sv2 ) at peripheral cholinergic nerve terminals , the receptor - toxin complex is internalized into a membrane - bound compartment that undergoes a drop in ph .
conformational changes occur that allow the insertion of the h - chain into this compartment 's membrane . as a result ,
the disulfide bond that links the l and h chains is reduced , an ion channel is formed , and the presumed proteolytic active moiety , the l - chain , is translocated into the neuroplasm . when the type a toxin substrate , snap-25 , is selectively cleaved , synaptic vesicle - mediated neurotransmission is blocked that could eventually lead to fatal paralysis . since the 1980s , the therapeutic potential of this toxin has been exploited .
extraocular muscles have been injected with the partially purified neurotoxin as an adjunct or alternative to surgical correction in treating strabismus [ 7 , 8 ] .
the chemodenervation effects of this most poisonous of poisons have been used to relax hyperkinetic striated muscle groups to diminish the effects of dystonia and related diseases .
food and drug administration ( fda ) for the following indications : strabismus , blepharospasm , cervical dystonia , upper limb spasticity , maxillary hyperhidrosis , chronic migraine , and urinary incontinence .
these indications along with the temporary enhancement in the appearance ( cosmesis ) with botox cosmetic of moderate to severe wrinkles in adults [ 13 , 14 ] introduces the theme of muscle immobilization in terms of a desired therapeutic outcome .
advantage has also been taken of this toxin 's chemoimmobilization property to improve the healing of wounds .
in contrast to the relatively vast amounts of information regarding this toxin 's structure and mechanism of action , the newer , off - label uses for botulinum toxin have been less extensively reviewed . to gain further insight regarding the scope of these efforts ,
we have gathered and examined biomedical research articles by conducting systematic searches of the relevant literature in pubmed , in a manner similar to that of steele and madoff .
we have examined studies that range from descriptive observations to randomized controlled clinical trials to obtain more information about the components and processes involved in wound healing and the related time courses of action of botulinum toxin a. the processes observed clinically on the wound healing effects of the type a toxin are at an early stage of our understanding .
this proposal is substantiated by evidence - based reviews that critically evaluate this toxin 's effects with different indications [ 17 , 18 ] .
we previously noted [ 19 , 20 ] that only a few clinical studies have focused on kinetic analyses .
constructing even partial models for the clinically observed effects by this toxin remains a challenge .
to advance our understanding , we have selected some of those clinical studies that have examined the timing of this toxin 's effects .
botxminer , the botulinum reference tool of clostridial neurotoxin citations in entrez - pubmed / medline , was initially used to search in article titles , abstracts , and mesh headings for the words wound and heal or healing . a more extensive list of 29 wound - related keywords
was then generated : anal , angiogenesis , collagen , cytokine , fibroblast , fibroblastic , fibrosis , fissure , flap , glycosaminoglycan , heal , healing , hemorrhoid , hemorrhoidectomy , hypertrophic , incision , inflammation , inflammatory , keloid , lesion , repair , scar , scarring , sphincterotomy , surgical , tendon , tensile , ulcer , and wound .
this controlled vocabulary was used in the batch mode to search botxminer for additional related citations .
another set of filter terms were used to find time - course - related information about this toxin .
this set included 26 terms : clearance , day , decay , decline , delay , diffusion , duration , follow up , frequency , hour , hr , interval , kinetic , latency , minute , month , onset , period , persistence , recurrence , repeat , resistance , sec , time , week , and year . within the two lists in the batch matrix search , ( a1 , a2 , , am ) and ( b1 , b2 , , bn ) , the terms undergo the or operation ( ) which can be represented in a general form of ( a1 or a2 or , etc . ) .
in addition , the lists are combined with the and operation ( ) which results in a batch query that can be represented by
( 1)(a1a2am)(b1b2bn ) .
summaries from the two sets of terms were returned by botxminer in the form of tables , histograms , and lists .
additional keywords and phrases within the more than 70 downloaded text files were automatically searched with file search assistant ( v. 3.1 , 2009 , aks - labs , raleigh , nc , usa ) . data from the clinical literature that were analyzed were fitted to an exponential function
( 2)y = y0+(a)exp(kdecayt ) ,
where y is the cumulative number of patients who are free of symptoms at time = t , y0 is the cumulative number of patients who are symptom - free at t = , a is a preexponential constant , and kdecay is the rate constant for the decay of this response .
chicago , ill , usa ) was used to conduct a least - squared fit for the values of y0 , a , and kdecay .
this equation is commonly used for simulating the decay rate of a reactant from a single model compartment .
the fda has approved generic , nonproprietary names for commercial formulations of botulinum toxin [ 2224 ] . for botulinum toxin type a , botox ( allergan , calif , usa ) is onabotulinumtoxina , dysport ( ipsen biopharm limited co. , uk ) is abobotulinumtoxina , and xeomin ( merz pharma gmbh & co kgaa .
, germany ) is incobotulinumtoxina . for botulinum toxin type b , myobloc / neurobloc ( solstice neuroscience , inc .
, pa , usa ; eisai ltd . , uk ) is now rimabotulinum toxin b .
the changes in nomenclature emphasize the different potencies and the noninterchangeable unit dosages of these distinct brand name products .
as reviewed by alberto , these distinctions in names emphasize the differences in manufacturing and formulation techniques that may contribute to differences in the pharmacokinetics , efficacy , safety , and antigenicity among these products . in the present paper , partially purified toxin with nontoxic or accessory proteins
is referred to as botulinum toxin type a to distinguish it from botulinum neurotoxin a ( bont / a ) , the pure holotoxin .
the lists of selective keywords are not intended to be exhaustive but serve as a starting point for the present work .
additional terms , such as proctology and coloproctology , can be used in more comprehensive studies .
verification confirms that , for example , the equations being coded are producing the correct calculations .
software can be validated when it can model the results that best fit existing data , and is subsequently reinforced by further data obtained experimentally . for kinetically related clinical problems , the underlying processes that need to be included are still uncertain .
furthermore , mathematical models are not designed to replace validation by basic research experiments or clinical observations .
rather , models are meant to enhance validation procedures by providing stimuli for new ideas , hypotheses , and perspectives on the problems being examined .
a preliminary search of botxminer , using the query wound and ( heal or healing ) for years 19802010 , returned over 150 citations about half of which were true positives due to the large number of references related to tetanus ( false positives ) . from the list of true positive citations ,
a variety of indications were found , in which botulinum toxin a has been used for wound healing and related conditions .
these examples included experimental cutaneous scars in animal models [ 15 , 28 ] and in clinical studies : chronic anal fissures [ 29 , 30 ] , cleft lip surgical repair , traumatic head lacerations or elective excisions of forehead masses , focal fold granuloma , hypertrophic scarring , pressure ulcers [ 35 , 36 ] , raynaud 's phenomenon ( vasospastic ischemia of the digits , digital ischemia , including chronic ulcers ) , and self - mutilation injuries in lesch - nyhan syndrome .
another healing application of botulinum toxin a , referred to as protective ptosis , is used against persistent corneal ulcers , burns , and other ophthalmic - related problems [ 39 , 40 ] . conducting a matrix batch search in which
tetanus was filtered out , using 26 time - related terms along with the 29 wound healing - related terms , yielded 671 unique citations . from this filtered output , we concentrated on references dealing with wound conditions and indications in which botulinum has been used for therapeutic purposes in which some mechanistic , dosage , and/or kinetic information was also available ( methods , figure 1 ) .
the normal wound - healing process has been described as being comprised of four overlapping phases : haemostasis , inflammation , tissue proliferation , and remodeling .
if any of these processes are disrupted , healing is impeded leading to a chronic wound state .
the interference by botulinum toxin in reducing muscle movement has helped to define healing phases further .
for example , a vicious cycle involving inflammation , pain , and muscle spasm was first noted to be the underlying cause for the development of chronic anal fissures [ 29 , 42 ] .
subsequently , low blood flow and ischemia were added to this cycle [ 4345 ] .
additional components of the healing process have been identified for other conditions as presented in this section .
scar formation is a hallmark of wound healing , and it usually causes significant physical , psychological , and cosmetic problems .
hypertrophic scarring is a common , refractory dermal disease that is manifested by the abnormal appearance of wound healing which can be the result of different types of injuries [ 34 , 46 , 47 ] . in the study by xiao et al .
, 19 patients were treated once a month over 3 months with 1.835 u botulinum toxin a ( hengli / cbtx - a , lanzou biochemical co. , china ) .
scores were assigned before treatment for the associated erythema , pliability ( lesion softening ) , and the severity of itching . after a 6-month followup of the 19 patients participating in that study , 15 gave an overall assessment of their lesion improvement as good , and seven others rated their improvement as excellent .
some critical comments provided by the authors included the lack of control subjects , the study was not double blinded , and a small patient population size .
finally , there was only a relatively short follow - up time of 6 months so that no determination of the total time course of toxin action could be established .
a number of quantitative parameters may be accessed to evaluate the effectiveness of wound healing by botulinum toxin a. increased metabolic activity and inflammation during the healing process induce muscle contractions around the edges of the skin wound [ 32 , 46 ] .
the major role of botulinum toxin a in this healing process is to prevent the repeated , small contractions that produce microtraumas near the hypertrophic scar and thereby decrease the tensile force ( muscle activity ) during scar formation .
traditional surgical techniques that align incisions along langer 's lines do not prevent repeated contractions .
the development of fibrosis also involves the deposition of extracellular collagen and glycosaminoglycans that can cause the scar to hypertrophy , invert , and become hyperpigmented resulting in poor color matching of this tissue with the neighboring skin [ 15 , 28 ] .
other parameters for wound healing include size of wound , amount of and infiltration of inflammatory cells , blood vessel proliferation , and wound thickness .
additional cellular and molecular mechanisms of healing by the formation of scar tissue ( traumatic cicatrisation ) are beginning to be elucidated . transforming growth factor 1 ( tgf- 1 )
is a fibrotic cytokine that stimulates cellular growth , differentiation , and adherence and leads to the collagen deposition .
this cytokine initiates these processes by extracellularly binding to a coupled pair of serine - threonine kinases . on binding , one receptor recruits and phosphorylates the other
this signaling pathway eventually stimulates transcription of the collagen gene and the formation of hypertrophic scars . because human fibroblasts derived from hypertrophic scars overexpress and secrete tgf-1 , another wound - healing effect of botulinum toxin a has been speculated to be inhibiting the secretion of tgf-1 .
similarly , suramin , an antifibrotic polysulfonated naphthylurea compound , has been reported to promote wound healing by antagonizing tfg-1 in muscle - derived fibroblasts .
the circulatory system is also affected by the apparent ability of botulinum toxin to enhance wound healing . using a rat model for wounds ,
yoo 's group observed that pretreatment with botulinum toxin a increased dorsal skin flap survival and concluded that this process was caused by increased perfusion .
because this toxin inhibits secretion of norepinephrine from sympathetic vasodilator and vasoconstrictor neurons [ 51 , 52 ] , the effect of botulinum toxin a may involve increased perfusion by decreasing sympathetic vasoconstriction in the skin flaps , thus promoting skin flap survival .
the protective effects of botulinum toxin - induced ptosis have been used for the conditions of recalcitrant corneal ulcers and other surface disorders as an alternative to the surgical practice of partially sewing the eyelids together ( tarsorrhaphy ) .
this secondary healing effect is produced when botulinum toxin a is injected into eye muscles , typically the levator palpebrae superioris ( lps ) muscles .
this therapeutic application has been the subject of studies that have also generated kinetic data . from an open - label , multicentered study with 16 ophthalmic patients who received 5 u botox in the lps muscle , the time to suitable
ptosis was 4.0 0.5 days ( mean se , range : 28 days ) , and the duration of this ptosis was 46.0 12 days ( 1206 days ) . a similar number of patients who received a single , lower dose ( 2.5 u ) had a comparable mean time to ptosis ( 3.6 days ) and a shorter , mean duration ( 16 days ) .
although statistical analyses with more patients are required to make more definitive conclusions , these trends are not unexpected but are , nevertheless , remarkable because a twofold change of a low dose apparently resulted in appreciable differences in duration .
chronic anal fissure ( caf ) is a painful condition caused by spasms of the internal sphincter smooth muscles .
the traditional surgical approach has been sphincterotomy that can result in the adverse effect of incontinence .
traditional nonsurgical approaches that have been used include sitz bath , topical anaesthesia , nitroglycerin , isosorbide dinitrate , nifedipine , diltiazem , l - arginine gel , hyperbaric oxygen , and botulinum toxin a .
the first use of botulinum toxin as a medical alternative for caf was conducted by jost and schimrigk . for this indication , we chose what has been described as one of the longest follow - up studies using botulinum toxin ( type a ) in the treatment of caf .
we analyzed data for the time course of recurrence of caf symptoms in patients in a 42-month follow - up study [ 55 , table 1 ] .
as illustrated in figure 3 , at the beginning of the followup ( at t = 0 ) , all 53 patients were symptom - free for 6 months after one to two injections of 1020 u botox .
most patients ( 31 of 53 ) did not undergo recurrence indicating that they remained healed during this follow - up period . from the nonlinear fit of these data to ( 2 ) , the fitted values sems are y0 = 29.2 1.2 , a = 24.4 1.3 .
the kdecay ( decay rate constant ) is 7.08 10 1.07 10 month , 1.64 10min ( see below ) , or 0.85 years ( see table 1 ) .
the value of 1/kdecay or is 14.1 months , and the corresponding value of t1/2 ( = ln2 ) is 9.8 months or 294 days .
the long - term success rate was only 31/53 ( 58% ) or a 42% rate of recurrence . from arroyo 's group ,
recurrence rate of 12% occurred initially , and a rate of 53% at the 3-year follow - up point .
bilateral fissurectomies of the internal anal sphincter combined with toxin injections have been reported to have a high success rate although the follow - up time in that study was limited to 1 year .
also , it was not determined whether surgery itself produced a similar rate of healing .
an interpretation of the single exponential decay curve during the 42-month followup ( figure 3 ) is that it reflects a zero - order elimination or inactivation step of the persistent , intraneuronally located toxin .
this step is described by the rate constant of decay , kdecay , in figure 4 and ke in a previous publication .
the table summarizes the processes displayed in figure 4 and highlights the recurrence of symptoms that may be used to gauge the slow elimination or inactivation of the type a botulinum toxin .
notably , this rate may be comparable to the slow rates of healing of some chronic wounds .
alternatively , the long - lived toxic effect may be related to the rate of restoration of intact snap-25 intracellular levels or a combination of a slow degradation and a persistent inhibitory action of the snap-25 bont / a cleavage product .
the value of t1/2 of 294 days is comparable to the recurrence times of 444 132 days ( mean s.d . ;
range 270718 days ) for achalasia patients after receiving a single injection of 80 u botox . on the other hand ,
our calculated decay rate constant of 1.64 10min is about 1000 times slower than the estimated rate constant for decay of efficacy ( 1.1 10min ) in a single dystonic patient [ 58 , 61 , 62 ] .
this difference may be due to several factors , among which are the different patient populations , the muscles being injected , the conditions being treated , and the assessment methods .
while the above studies show encouraging trends in support of using botulinum toxin a in wound - healing paradigms , additional studies are necessary .
overall , more prospective clinical studies of these treatments with botulinum toxin a are needed .
evidence from blinded , randomized , placebo - controlled , multicentered studies will help determine if these toxin treatments have significant benefit and if the minimal adverse reactions can be sustained .
future trials should also use larger populations of more homogeneous ( standardized ) patients and control subjects , plan to examine long - term outcomes , and conduct cost - benefit analyses .
although randomized controlled trials are considered the gold standard of clinical research , assessing them using criteria for standardizing phase iii trials remains a substantial challenge .
moreover , there is also a need for additional controlled studies to clearly establish an advantage of botulinum products over other methods .
an outcome of one of these analyses was the low probability that type a botulinum toxin or calcium channel blockers were found to be more effective in treating caf than nitroglycerin ointment in 182 patients .
's analysis which determined that for 279 caf patients the traditional surgical procedure of lateral internal sphincterotomy ( lis ) was more effective than botox in healing chronic anal fissure .
while lis produced a higher rate of minor anal incontinence , botulinum toxin was associated with a higher rate of recurrent disease . for those patients who had a high risk of incontinence , local injection of the toxin
computational modeling and simulation studies at different levels of granularity ( i.e. , detail ) should also be beneficial .
starting with existing minimal kinetic models [ 19 , 66 ] , dose - dependent kinetic models could be developed to predict the time course ( onset , duration , and recurrence of symptoms ) and the extent of botulinum toxin a 's effectiveness .
kinetic models could help to identify what research gaps exist and which ones can be experimentally or clinically resolved .
one gap that could be experimentally verified is to determine if the intracellular diffusion of botulinum toxin a is influenced by other coinjected materials , for example , epinephrine and local anesthetics ( lidocaine , xylocaine ) , compounds that have been considered in controlling local diffusion and predicting the extent of this toxin 's paralytic effect [ 15 , 32 ] . as more realistic physiological - pharmacological models are developed , more free parameters and more sources of error , assumptions , and caveats will need to be evaluated . potentially confounding
factors associated with wounds include cell viability , alkali conditions , the generation of reactive oxygen species , and inflammation that is related to low blood flow and ischemia .
changes in the ion flow through transmembrane channels and in metabolism could also have roles in wound healing .
another confounding factor is the well - known , persistent muscle weakness ( up to 5 years ) that results from protracted patient immobilization due to critical illness polyneuropathy or myopathy [ 68 , 69 ] .
this persisting weakness and residual neurologic deficits are likely due to denervation , combined with catabolic muscle wasting and potential myopathic changes .
it remains for future studies to differentiate these physiological deficits from botulinum toxin - induced effects in either a botulism patient or with therapeutic treatments with this toxin .
the tissues involved in the healing process in cytoskeletal architectures and in membrane structures associated with various organs also need to be considered .
neurophysiological abnormalities , such as transient denervation - induced muscle fibrillations and presynaptic alterations producing muscle fasciculations , may also need to be considered . other complicating factors in the healing process could also involve the biomechanical dynamic properties of soft tissue ( e.g. , stiffness ) in response to stress and strain , and tissue anisotropy ( directionality of nonhomogeneous material ) .
this succinct review examines the soft tissue wound - healing properties of botulinum toxin . when viewed from the perspective of treating neurologic and other disorders
, it is noteworthy that the efficacy of this toxin is predicted to be transient as the toxin 's effect wanes , while for wound healing , a permanent resolution is expected .
further , with respect to wound healing , the concept is described that for some lesions , botulinum toxin interferes with the vicious cycle of muscle spasm , pain , inflammation , decreased blood flow , and ischemia .
a reaction pathway scheme is outlined to illustrate botulinum toxin 's involvement in stopping the vicious cycle . a minimal kinetic scheme for healing chronic wounds is also presented that includes different macroscopic states of soft tissue conditions ( normal , initial wound , and chronic wound ) , and quantitative estimates for the relevant rate constants are provided .
a definitive validation of the results , that is , the minimal kinetic model for predicting the beneficial effects of type a toxin , awaits additional clinical data .
perhaps the most useful outcome is that our kinetic model is capable of identifying a measurable gap ( decay rate of toxin 's effect ) in our attempt to comprehend how complex , interacting biological systems respond to environmental stressors . | a relatively new
approach in the treatment of specific wounds in
animal models and in patients with type a
botulinum toxin is the focus of this paper .
the
indications or conditions include traumatic
wounds ( experimental and clinical ) , surgical
( incision ) wounds , and wounds such as fissures
and ulcers that are signs / symptoms of disease or
other processes .
an objective was to conduct
systematic literature searches and take note of
the reactions involved in the healing process
and identify corresponding pharmacokinetic data .
from several case reports , we developed a
qualitative model of how botulinum toxin
disrupts the vicious cycle of muscle spasm ,
pain , inflammation , decreased blood flow , and
ischemia .
we transformed this model into a
minimal kinetic scheme for healing chronic
wounds .
the model helped us to estimate the rate
of decline of this toxin 's therapeutic
effect by calculating the rate of recurrence of
clinical symptoms after a wound - healing
treatment with this neurotoxin . |
previous studies have demonstrated the efficacy of transversus abdominis plane ( tap block ) in providing adequate postoperative analgesia for up to 24 h after lower abdominal surgery .
laparoscopic cholecystectomy is associated with moderate postoperative pain in the early postoperative period , and multimodal analgesia approach may be necessary . for
day care surgical procedures , pain and issues with surgery and anesthesia may prevent same - day discharge .
postoperative pain treatment with opioids increases the likelihood of side effects such as nausea and sedation .
recently the surge in use of tap block , which may provide up to 24 h of analgesia , promises to be an interesting analgesic option .
however , it has been questioned whether a tap blockade at the umbilical level can provide analgesia in an upper abdominal surgical procedure such as laparoscopic cholecystectomy .
conflicting results are evidenced from studies on tap versus local anesthetic ( la ) to treat postoperative pain for laparoscopic cholecystectomy .
a recent review showed no significant difference in clinical outcomes between these two techniques but tap block reduces pain scores .
the aim of this study was to investigate the effect of la infiltration and tap block on postoperative pain , opioid consumption and subsequent side effects .
the secondary outcome was quality analysis by doing patient satisfaction and cost in these two methods .
the study was approved by the ethics committee and registered at the clinical trial registry .
the data were collected from hospital theatre database and medical records for the years 2011 - 2012 .
the sample included day surgery patients who underwent a laparoscopic cholecystectomy and who met the following inclusion and exclusion criteria .
aged 18 - 84 years , had laparoscopic cholecystectomy via day surgery during december 2011-may 12 , receiving either la infiltration with 20 ml 0.25% bupivacaine or bilateral subcostal tap block with 20 ml 0.5% ropivacaine on each side .
the primary outcome was pain scores and fentanyl analgesia used as indicated in the recovery record .
as per our institutional protocol , pain management includes fentanyl 20 mcg/3 min bolus for 15 min then oxycodone 5 - 10 mg .
the pain scores recorded were on numerical rating scale , many times but two documented scores , the first on arrival to the recovery and second after 1 h in recovery .
the quality of pain provided by each technique used was assessed 30 - 180 days after surgery by telephone using a likert satisfaction scale ( satisfaction score rating on scale of 1 - 4 as follows : completely relieved ; 2 .
the student 's t - test or wilcoxon ( mann - whitney ) was used to assess group differences .
dichotomous measures were reported as percentages and were assessed using the pearson 's chi - square statistic or fisher 's exact test .
aged 18 - 84 years , had laparoscopic cholecystectomy via day surgery during december 2011-may 12 , receiving either la infiltration with 20 ml 0.25% bupivacaine or bilateral subcostal tap block with 20 ml 0.5% ropivacaine on each side .
the primary outcome was pain scores and fentanyl analgesia used as indicated in the recovery record .
as per our institutional protocol , pain management includes fentanyl 20 mcg/3 min bolus for 15 min then oxycodone 5 - 10 mg .
the pain scores recorded were on numerical rating scale , many times but two documented scores , the first on arrival to the recovery and second after 1 h in recovery .
the quality of pain provided by each technique used was assessed 30 - 180 days after surgery by telephone using a likert satisfaction scale ( satisfaction score rating on scale of 1 - 4 as follows : completely relieved ; 2 .
the student 's t - test or wilcoxon ( mann - whitney ) was used to assess group differences .
dichotomous measures were reported as percentages and were assessed using the pearson 's chi - square statistic or fisher 's exact test .
120 patients assessed for the year 2012 . of these , 39 patients did not fulfil the inclusion criteria , and 30 had incomplete data .
the remaining 51 patients were included in the analysis , with 19 patients in the tap group , and 29 in the la group .
the groups did not significantly differ in terms of age , sex , weight or postanesthesia care unit time [ table 1 ] .
demographic characteristics and pacu time there were no significant differences between the la and tap groups with respect to postoperative pain scores [ table 2 ] ( p = 0.31 ) and patient likert scale of 1 - 4 satisfaction scores [ table 3 ] ( p = 0.36 ) . majority of them were scores of 1 and 2 .
mean fentanyl consumption in the recovery room was significantly higher in the la group compared to tap group ( p = 0.0079 ) [ table 2 ] although recovery room oral oxycodone consumption did not differ between the groups [ table 2 ] ( p = 0.66 ) .
consumables cost for the tap block were more than three times that for la group [ table 4 ] .
in this study , the performance of tap block was equivalent to la infiltration technique at least in terms of pain scores and satisfaction scores . mean fentanyl consumption in the recovery room was significantly higher in the la group when compared to tap group though the oral oxycodone consumption was not significant .
these observations are consistent with the findings reported elsewhere . for instance , ortiz et al . found that bilateral ultrasound - guided tap block was equivalent to la infiltration technique in a prospective randomized trial in patients undergoing laparoscopic cholecystectomy .
we found the fentanyl consumption in the recovery room was significantly lower amongst patients in the tap group .
however , it is possible that fentanyl consumption represents a more objective marker of postoperative pain than the pain scores on the hospital record , and if this were the case , then the superiority of tap block over la would be clearly demonstrated .
this is consistent with the recent review showing reduced pain and analgesia with tap block . in our study
studies report an additional reduction in use of intraoperative opioids if tap block is instituted preoperatively .
the preoperative tap block resulted in a 63% reduction of intraoperative analgesic requirement and a 54% reduction in early postoperative analgesic requirements in one study , while a second study reported 44% reduction in intraoperative opioid consumption ( remifentanyl ) but no clinically significant reduction in postoperative opioid consumption .
transversus abdominis plane block results in substantially higher consumables costs when compared to la infiltration .
however , other costs have not been considered , including theatre and surgeon cost and costs associated with inpatient admission resulting from persisting pain .
it 's not clear whether the cost differential would persist once these additional costs were included .
in previous studies where tap and la were compared using either posterior or subcostal approach tap blocks , sensory assessment was demonstrated .
the former approach may not be sufficient to reach sensory dermatomes to cover analgesia for portal incisions in the subcostal regions .
although the latter approach covers analgesia , which had reduced recovery time and pain score , but the studies used other oral analgesics like tramadol and codeine . furthermore nausea and vomiting ( nv ) were not recorded despite the fact that these may contribute to recovery discharge time in the tap group .
we recommend a prospective randomized study comparing posterior tap or rectus sheath block and subcostal tap block with la infiltration technique to determine its efficacy .
transversus abdominis plane block is compartmental and the dose required was more in comparison to la group , hence dose variability may cause a considerable difference .
in addition surgeon practice is limited to bupivacaine and anesthetist doing tap block using ropicavcaine , hence drug variation can differ .
the time duration for la infiltration and tap block were not available in the medical records .
the pain scores were only two readings taken and dynamic pains scores ( coughing ) would have been valuable . there was no documentation of sensory assessment that may be vital in assessing whether the block was successful block rather than just assuming it was working .
satisfaction scores assessment time varied from 30 to 180 days , which may have a chance of bias .
the performance of the la technique was comparable to the bilateral tap block in respect to pain scores amongst patients presenting for day surgery laparoscopic cholecystectomy . | background and aims : postoperative pain for day surgery laparoscopic cholecystectomy has traditionally been managed with the surgeon infiltrating the wound with local anesthetic ( la ) . however ,
transversus abdominis plane ( tap ) block has recently been used , although its superiority over la remains uncertain .
the primary aim was to compare la and tap block pain scores and analgesia used .
the secondary aim was to assess satisfaction score and cost.material and methods : this retrospective study was commenced after ethics committee approval and anz clinical trial registry ( actrn : 12612000737831 ) .
the data were collected from the theatre database and medical records of patients presenting for day case laparoscopic cholecystectomy .
the sample included patients who received either bilateral port site la infiltration with 20 ml of 0.25% bupivacaine or bilateral tap block with 20 ml of 0.5% ropivacaine and fentanyl postoperative pain protocol .
the patients with incomplete medical records were excluded as were those admitted to an inpatient ward . demographics and clinical characteristics were obtained from the hospital record along with pain score and postsurgery analgesia use .
postoperative pain satisfaction scores were collected by telephonic interview 30 - 180 days postsurgery.results:of 51 patients analyzed , 19 were in tap group 29 in la group .
there were no significant differences between the la and tap groups with respect to postoperative pain scores ( p = 0.31 ) or patient satisfaction scores ( 1 and 2 + ) ( p = 0.36 ) . however , fentanyl consumption in the recovery room was significantly lower in tap group ( p = 0.0079 . ) .
the consumables cost were > 3 times higher in the tap when compared to la group.conclusion:the performance of the tap block with respect to pain management was comparable to la .
however , la remains more cost effective . |
there are numerous sources of suspended particulate matter ( pm ) including industrial sources , automobile traffic , natural disasters , such as forest fires and volcanic eruptions , and local sources generated either in the home or workplace [ 13 ] .
urban air pollution originates from a variety of sources , of which the combustion of fossil fuel products is the principal source .
air pollutants can be classified by their source , chemical composition , size , mode of release ( gaseous or particulate ) , and space ( indoor or outdoor ) .
epidemiological studies show that air pollution exposure positively correlates with admissions for pneumonia , asthma , and chronic obstructive pulmonary disease ( copd ) .
of all the pollutants , inhalable particles ( pm10 ) showed the strongest association with adverse respiratory health effects .
in addition , data from large population cohorts have indicated an association between exposure to pm and cardiovascular morbidity and mortality [ 710 ] .
mechanistically , this is thought to be due to the systemic inflammatory response induced by exposure to pm air pollution .
this concept is supported by studies showing a positive association between long - term pm exposure and hematological markers of inflammation and diseases such as diabetes mellitus .
inhalation of air pollution particles induces a local response in the lung that is initiated by alveolar macrophages ( ams ) and airway epithelial cells .
the macrophages are several times more potent in producing proinflammatory mediators that contribute to the local inflammatory response in the lung but also contribute to the subsequent systemic inflammatory response .
the systemic inflammatory response is characterized by mobilization of inflammatory cells from the bone marrow into the circulatory system , followed by their activation , as well as the production of acute phase proteins by the liver , and an increase in circulating inflammatory mediators . in this review ,
the primary role of am is to keep the air spaces clear by removal of all foreign materials via phagocytosis .
experiments from our laboratory have shown that ams exposed to atmospheric particulate matter are able to phagocytose these particles in vivo and in vitro [ 13 , 14 ] .
nonbiological particles lack specific opsonins preventing them from classic opsonin - dependent phagocytosis . despite the absence of specific opsonins , am can phagocytose unopsonized environmental particles .
the class a scavenger receptor ( sr - a ) and the macrophage receptor with collagenous structure ( marco ) are considered to be the two major receptors for unopsonized particle phagocytosis by am [ 17 , 18 ] , and a deficiency in scavenger receptor function results in reduced uptake of environmental particles by am .
toll - like receptors ( tlrs ) are sensors that directly recognize molecules from microbes .
they are essential for initiation of the innate immune response , when interacting with pm , and also play a role in sustaining and regulating the adaptive immune response to pm .
ambient pm contains small amounts of microbial materials such as lipopolysaccharide ( lps)/endotoxin [ 19 , 20 ] , beta - glucan , bacteria , and fungal spores that are thought to be the mechanism by which tlrs engage in processing pm . among the tlrs identified in humans , tlr4 and tlr2
toll - like receptor 4 initiates a signaling cascade in response to lps present in the outer membrane of gram - negative bacteria , while tlr2 initiates signals in response to zymosan ( beta - glucans ) and peptidoglycan of gram - positive bacteria .
furthermore , microorganisms attached to pm can be opsonized by specific opsonins ( such as immunoglobulin fc receptors and complement receptor 3 ) that allow am to phagocytose the particles via an opsonin - dependent pathway .
particulate matter is classified according to aerodynamic diameter into pm10 ( coarse particles , median aerodynamic diameter 2.510 m pm2.5 ( fine particles , median aerodynamic diameter < 2.5 m ) , and ultrafine particles ( ufp ) median aerodynamic diameter < 0.1 m ) .
pm10 particles are derived predominantly from abraded soil , road dust , construction debris , and oil combustion products with bioaerosols such as fungi , bacteria , endotoxins , and pollen , while pm2.5 and ufp are primarily derived from direct emissions from combustion processes such as vehicle use of fossil fuel products , wood burning , and coal burning .
although a considerable amount of data implicate pm10 and pm2.5 in adverse health effects [ 2426 ] , much less is known about the risks of ufp .
in addition , several studies have shown that pm2.5 and ufp have the strongest association with adverse cardiovascular adverse effects [ 27 , 28 ] , which is a direct consequence of the systemic response induced by these particles [ 29 , 30 ] .
alveolar macrophages exposed to smaller pm that have the ability to penetrate deep into the lungs significantly contribute to the systemic inflammatory response . upon contact with particulate pollutants ,
the capability of inducing apoptosis and inflammation varies with different particle size and concentration . in vitro studies
have shown that macrophages do recognize the size and shape of their target pathogens ; therefore , their response against various particle sizes may be different .
it is generally thought that the larger surface area of pm2.5 and ufp per unit concentration of pm allows more opportunity for cellular interaction and a downstream biological response . on the contrary , in vitro studies comparing the effects of the coarse and fine fraction of pm10 showed stronger proinflammatory effects for the coarse particles [ 33 , 34 ] .
in addition to pm size and concentration , particle composition has also been reported to impact pm toxicity .
the toxicity of pm may stem from their metal content , adhered organic compounds , or other biological components such as lps .
. showed that coarse pm induced a greater inflammatory response than fine pm in rats and suggested that , in these larger particles , toxicity is due more to their biological components , such as endotoxin , than their metal content . in other studies , no apparent relationship could be established between pulmonary injury and the concentration of ambient particles or their elemental components such as sulfate ( s ) , zinc ( zn ) , manganese ( mn ) , iron ( fe ) , and copper ( cu ) .
diesel exhaust particles ( deps ) without their organic constituents were no longer able to induce apoptosis or generate reactive oxygen radicals in murine and human macrophages in vitro .
diesel exhaust particle is a major component of urban pm10 pollutants , which comprise 40% of total pm10 levels in los angeles .
the water - soluble fraction of pollutant particles and individual soluble metals such as vanadium ( v ) , nickel ( ni ) , and fe did not induce apoptosis in human ams .
in addition to these reports that emphasize the toxicity of organic components in pm , nonorganic components such as metals have also been implicated in the pathogenesis of particulate - induced pulmonary inflammation .
vanadium ( v ) , bromine ( br ) , lead ( pb ) , and organic carbon had a strong association with pulmonary inflammation .
stone particles of varying composition ( mylonite , gabbro , feldspar , basalt , and quartz ) induced different cytokine responses in rat ams . because ambient particles contain many other nonleachable and leachable components , further studies are needed to identify the toxicity of different particle components .
in addition to pm , gaseous pollutants such as ozone also have inflammatory effects on the respiratory tract and am [ 4245 ] .
ozone exposure induces the release of cytokines and fibronectin by am , increases am recruitment into asthmatic airways , and increases the eosinophilic airway response .
nitrogen dioxide ( no2 ) is a precursor to photochemical smog , and its major effect on health as an outdoor pollutant is likely through the formation of ozone .
recent epidemiologic studies conducted worldwide have provided valuable insight into the associations between sulfur dioxide ( so2 ) , no2 , and carbon monoxide ( co ) exposure and increases in cardiopulmonary mortality , such as respiratory and cardiovascular hospital admissions , emergency admissions caused by stroke ( no2 ) , and myocardial infarction ( no2 and co ) [ 4951 ] . in these studies , no2 inhibited ams from playing an immunosuppressive role .
alveolar macrophage phagocytosis was significantly suppressed following coexposure of fine carbon particles and so2 . because air pollution contains both , the contribution of these gaseous pollutants in modulating the response of the am to pm is complex , poorly understood , and still an area of active investigation .
alveolar macrophages are one of the most potent producers of proinflammatory mediators in the airways and lung .
studies from our laboratory have shown that human ams exposed to urban pm ( ehc-93 ) phagocytosed these particles in vitro ( figure 1 ) [ 13 , 54 ] , and , ex vivo , they produced tumor necrosis factor alpha ( tnf- ) in a dose - dependent fashion following pm exposure .
in addition , these ams produce an array of proinflammatory mediators including acute response mediators such as interleukin ( il)-1 and il-6 as well as secondary mediators , such as il-8 , and granulocyte macrophage colony - stimulating factor ( gm - csf ) [ 11 , 13 ] .
interestingly , production of anti - inflammatory mediators such as il-10 was suppressed , suggesting that the am inflammatory response induced by pm is tipped toward a proinflammatory profile .
the inflammatory profile of mediators produced by bronchial epithelial cells exposed to pm is distinct from those of am .
alveolar macrophages are also more potent producers of the acute response mediators such as il-1 , il-6 , and tnf- than bronchial epithelial cells when exposed to the same dose of pm , suggesting that ams are the drivers of the proinflammatory response in the lung following inhalation of pm ( figure 2 ) . furthermore , instillation of supernatants from human ams incubated ex vivo with urban pm into rabbit lungs produced a systemic response similar to that produced by direct deposition of the same amount of pm directly into the lungs [ 57 , 58 ] , which implies that ams significantly contribute to the systemic inflammatory response generated following exposure to pm .
depending on the microenvironment , they undergo distinct activation programs , acquiring polarized phenotypes and different functional capacities that together provide an armamentarium that protects , repairs , and sometimes damages tissues [ 60 , 61 ] .
program , is induced by signals generated during th1-mediated immune responses such as interferon ( ifn)- and by exposure to components of pathogens such as bacteria [ 60 , 61 ] .
the m1 polarization response is characterized by upregulation of genes relevant to inflammation and cell - mediated immunity .
m2 polarization induced upon exposure to the th2 cytokines il-4 and il-13 ( referred to as alternative activation ) or immunoregulatory signals such as il-10 ( also called deactivation )
is highlighted by induced expression of receptors with scavenger functions , anti - inflammatory cytokines , and molecules implicated in tissue remodeling [ 60 , 61 ] .
although pm shares many of the same ingredients and characteristics as cigarette smoke , the m1 cytokines ( il-12 and ifn- ) are increased consistently in bronchoalveolar lavage fluid ( balf ) from pm - exposed animals [ 63 , 64 ] while the m2 cytokines ( il-4 , il-10 , and il-13 ) remain at lower levels .
our group previously showed that primary cultured human am , stimulated in vitro with urban pm10 , produced an array of cytokines without significantly increased levels of il-10 compared to nonstimulated am .
these reports suggest that pm skews the ams to an m1 profile rather than an m2 profile .
data from an influenza virus pneumonia model suggest that macrophage pro- and anti - inflammatory phenotypes are under tight control of nearby airway epithelial cells .
epithelial - macrophage crosstalk seems to be an important mechanism in keeping the balance between efficient host defense and excessive inflammation and injury during infection .
these responses ( m1/m2 switching ) of macrophages following pm exposure still need further investigation to assess what factors ( such as size or composition ) determine switching .
figure 3 shows how pm exposure could potentially inhibit macrophage switching and thus promote a proinflammatory state .
in addition to being key players in the innate immune response against microorganisms and in the initiation of adaptive immune responses , they are crucial for the clearance and processing of microorganisms , dead cells , and environmental debris in the lung tissue via phagocytosis .
in contrast to cells such as neutrophils , ams are long - lived and in general are resistant to apoptotic stimuli . following activation of am , by exposure to pm ,
for example , they either remain in the lung airways or tissues or are removed via the lymphatic system to regional lymph nodes .
several studies have demonstrated that exposure to ambient pm and diesel exhaust particles induced apoptosis in macrophages [ 37 , 71 ] .
phagocytosis of apoptotic cells ( efferocytosis ) by ams is involved in the regulation of the inflammatory response and maintenance of lung homeostasis by removing dead cells before the onset of necrosis .
alveolar macrophages are primarily responsible for removing and processing dead cells and debris in the airways , thereby reducing their inflammatory potential .
our group recently showed that 3-hydoxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase inhibitors ( statins ) enhance the phagocytic activity of ams and promote the clearance of pm from lung tissues .
promoting macrophage phagocytosis and efferocytosis could accelerate processing and clearance of pm particles from lung tissues and thereby reduce lung inflammation .
autophagy consists of the fusion of autophagosomes with lysosomes , forming autolysosomes and resulting in the breakdown of encapsulated materials to components that are then available for homeostasis .
intracellular nanoparticles may undergo autophagic sequestration , and autophagy dysfunction may play an important role in nanoparticle toxicity .
identified an autophagic defect in the ams of smokers and concluded that the decrease in the process of autophagy leads to impaired protein aggregate clearance , dysfunctional mitochondria , and defective delivery of bacteria to lysosomes .
exposure to ambient pm could decrease autophagy of am in a similar manner to smoking , but further studies are necessary to confirm this .
they sense , scavenge , and phagocytose pm and in the process they produce and release early response cytokines .
these cytokines stimulate neighboring airway and alveolar epithelial cells as well as tissue - resident macrophages in an auto- and paracrine manner to produce a variety of chemokines necessary to recruit other cells , such as polymorphonuclear leukocytes , to assist in processing and ultimately clearing foreign material .
human airway and alveolar epithelial cells are also capable of pm endocytosis and in the process they produce mediators such as gm - csf , il-1 , il-8 , and leukemia inhibitory factor ( lif ) in a dose - dependent manner at both the mrna and protein level when exposed to ambient particles .
coculture experiments of bronchial epithelial cells and am showed synergistic production of certain mediators such as il-1 , il-6 , and gm - csf . the increased il-1 production [ 54 , 76 ] is mediated by the nucleotide - binding domain and leucine - rich repeat protein 3 ( nlrp3 ) inflammasome that spreads the local inflammatory response by interacting with resident dendritic cells residing within or near the epithelium , initiating , and maintaining an adaptive immune response .
these studies demonstrate the importance of the interaction of ams with other lung cells in producing lung inflammation and possibly contributing to the systemic inflammatory response following pm inhalation .
after phagocytosis and internalization of pm , the organic components are digested by the endosome into peptide fragments that combine with the mhc class ii complex for presentation to cd4 + t cells , which are pivotal steps in cell - mediated and adaptive immunity .
expression of mhc in ams moderately increased in response to pm exposure in healthy human subjects .
pretreatment of pm with heat to degrade the organic component abolished the mhc class ii overexpression , which suggests that the organic component of pm is responsible for mhc class ii upregulation .
in addition to antigen - specific mhc and t - cell receptor interaction , t cells require a costimulatory signal to be fully activated .
these molecules are expressed on the cell membrane of antigen presenting cells and are upregulated by pm exposure .
together , these studies illustrate the crucial role that ams play in initiating the adaptive immune response to pm exposure .
however , the role of this adaptive response in the local lung and systemic inflammatory responses induced by pm exposure has not been well studied to date .
earlier studies implicate ams as key effector cells responsible for generating the systemic inflammatory response associated with exposure to air pollution [ 13 , 41 , 56 , 81 , 82 ] .
they produce a broad range of mediators , particularly il-6 , il-1 , mip-1 , and the hematopoietic growth factor gm - csf , when exposed to urban pm .
the importance of the am producing the mediators that elicit the systemic response is supported by studies showing a correlation between the amount of particles phagocytosed by am in the lung and the magnitude of bone marrow stimulation following pm exposure ( figure 4 ) .
humans exposed to an acute episode of air pollution , where the predominant pollutant was pm , showed increased levels of circulating cytokines such as il-1 and il-6 and signs of bone marrow stimulation reflected by an increase in circulating band cells counts .
these cytokines are similar to those produced by am exposed to pm both ex vivo and in vivo [ 13 , 84 ] , suggesting that these mediators produced in the lung enter the circulation and contribute to the systemic response associated with exposure to pm .
recent studies from our laboratory showed that mediators such as il-6 , produced in the lung from pm exposure directly , translocate into the circulation and , because the ams are the most prolific producer of these mediators following pm exposure , it is reasonable to postulate that ams are crucially important effector cells in generating the systemic inflammatory response induced by air pollution exposure .
several studies from our laboratory have shown that exposure to air pollution stimulates the bone marrow in humans and in animal models [ 11 , 14 , 5658 , 85 ] and promotes the release of both polymorphonuclear leukocytes and monocytes from the marrow .
the cytokine production by macrophages in the lung is of particular importance in inducing this systemic inflammatory response ; for example , gm - csf is a hematopoietic growth factor that stimulates granulocyte and monocyte differentiation and releases from the bone marrow , but it also activates circulating leukocytes and prolongs their survival in the circulation . furthermore , il-1 is one of the acute response cytokines that induces cytokine production by many cells , stimulates hematopoiesis , activates endothelial cells , is pyrogenic , and induces the acute - phase response .
in addition , stimulation of liver hepatocytes by il-6 produces acute phase proteins including c - reactive protein ( crp ) , fibrinogen , and antiproteases .
moreover , il-6 also stimulates hematopoiesis , specifically the production of platelets , and has a broad stimulating effect on b and t cells , as well as markedly accelerating the transit time of granulocytes through the bone marrow , releasing them into the circulation , and promoting their sequestration in microvascular beds .
collectively , gm - csf , il-1 , and il-6 have the ability to elicit a systemic inflammatory response characterized by an increase in circulating leukocytes and platelets by directly stimulating the bone marrow .
this bone marrow response elicited by pm exposure is thought to play a critically important role in the downstream adverse systemic health effects associated with exposure to air pollution , particularly the adverse effects on the heart and blood vessels .
numerous studies have shown an association between air pollution and increased cardiovascular morbidity and mortality [ 24 , 8891 ] .
the adverse cardiovascular health effects include hospital admissions and death from conditions such as acute myocardial infarction ( acute coronary syndrome ) , arrhythmias , and congestive heart failure .
these hospital admissions were shown to occur within hours of a spike in air pollution exposure .
elevated concentration of pm2.5 increased acute myocardial infarction within a few hours , which was confirmed by subsequent studies .
proposed that an increase in blood coagulability induced by deposition of particles in the lung is associated with an increase in cardiovascular deaths in susceptible individuals .
the systemic response induced by pm10 is characterized by activation of the acute - phase response , an increase in coagulation , the release of inflammatory mediators into the circulation leading to activation of the endothelium , and stimulation of the bone marrow causing the release of leukocytes and platelets .
these events may contribute to destabilization of atherosclerotic plaques , making them vulnerable for rupture and thrombosis and accounting for the increase in cardiovascular events associated with episodes of air pollution .
mediators such as gm - csf , il-1 , and il-6 produced by lung macrophages when exposed to ambient pm have the ability to elicit a systemic inflammatory response characterized by an increase in circulating leukocytes , platelets , and proinflammatory and prothrombotic proteins .
these mediators also have the ability to activate circulating leukocytes and the endothelium of the vascular bed to promote leukocyte - endothelial adhesion and migration , contributing to atherosclerotic plaque activation and instability .
our group showed that 4 weeks of exposure to ambient particles in watanabe hereditarily hyperlipidemic rabbits induced a systemic inflammatory response that included stimulation of the marrow and caused progression of atherosclerosis in both the aorta and coronary arteries , with phenotypic changes in atherosclerotic plaques characteristic of plaque vulnerability .
these observations have recently been confirmed by others using ambient pm2.5 and by our group , using deps in a mouse model . to determine the role of ams in these effects , studies by our group examined the relationship between the fraction of macrophages in the lung that had phagocytosed particles and circulating mediators strongly associated with cardiovascular disease in humans such as il-6 and found a positive association [ 84 , 98 ] .
in addition , our group showed an association between the extent of progression of atherosclerosis and features of plaque vulnerability and the fraction of ams that had phagocytosed pm ( figure 5 ) .
collectively , these studies strongly implicate the am in eliciting the systemic inflammatory responses induced by exposure to pm as well as the downstream adverse vascular effects of air pollution .
the studies mentioned earlier describe the effect of pm on ams ; however , it is understandable that there is marked interindividual variability in the response to pm .
exploration of genetic predispositions and epigenetic changes associated with ambient air pollution shows promising results .
polymorphisms in genes coding for glutathione - s - transferases ( gsts ) , which are enzymes responsible for the metabolism of reactive oxygen species , are correlated with the risk of lung diseases such as asthma when individuals are exposed to ambient air pollution [ 100 , 101 ] .
showed that single - nucleotide polymorphisms in tlr2 and tlr4 genes significantly modified the effect of pm2.5 on the incidence of asthma .
ambient air pollution can induce epigenetic changes such as dna methylation [ 103107 ] and there is further promise for genome - wide association studies ( gwas ) .
little is known about the effect of gene - environment interactions on am and more research in this field is required .
increasing evidence over the past 10 years has demonstrated that am plays a key role in local lung and systemic inflammatory responses induced by exposure to ambient pm . in the lung ,
am contributes to the magnitude and the nature of the inflammatory response by interacting with other lung cells such as bronchial epithelial cells and dendritic cells in an effort to process and clear the pm from the lung .
these macrophages also produce the mediators that are associated with the systemic inflammatory response induced by pm exposure , and recent studies support the concept that these systemic mediators translocate from the lung tissues into the circulation .
the adverse systemic health effects of exposure to pm , particularly the adverse cardiovascular effects , are strongly associated with the amount of pm phagocytosed by ams , underscoring the crucial role that the ams play in these adverse systemic responses of pm exposure .
therefore , it is reasonable to suppose that attenuating the local and systemic inflammatory responses of ams , induced by pm exposure , would be of benefit .
recent studies by our group have shown that statins reduce both local lung and systemic inflammatory responses induced by exposure to ambient pm in a rabbit model .
figure 6 shows a potential mechanism by which statins could alter key pathways to achieve these local and systemic anti - inflammatory effects . downregulating the inflammatory responses
further research surrounding gene - environment interactions in ams will contribute to understanding of interindividual variability and may assist in designing tailor - made therapies for air - pollution - related lung and heart diseases . | large population cohort studies have indicated an association between exposure to particulate matter and cardiopulmonary morbidity and mortality .
the inhalation of toxic environmental particles and gases impacts the innate and adaptive defense systems of the lung .
lung macrophages play a critically important role in the recognition and processing of any inhaled foreign material such as pathogens or particulate matter .
alveolar macrophages and lung epithelial cells are the predominant cells that process and remove inhaled particulate matter from the lung .
cooperatively , they produce proinflammatory mediators when exposed to atmospheric particles .
these mediators produce integrated local ( lung , controlled predominantly by epithelial cells ) and systemic ( bone marrow and vascular system , controlled predominantly by macrophages ) inflammatory responses .
the systemic response results in an increase in the release of leukocytes from the bone marrow and an increased production of acute phase proteins from the liver , with both factors impacting blood vessels and leading to destabilization of existing atherosclerotic plaques .
this review focuses on lung macrophages and their role in orchestrating the inflammatory responses induced by exposure to air pollutants . |
primary biliary cirrhosis ( pbc ) is an autoimmune disease in which the intrahepatic bile ducts are targeted by an immunemediated injury .
pbc is associated with a range of conditions , including sjgren 's syndrome in 70% of patients 2 , autoimmune thyroid disease in approximately 10% , and systemic sclerosis in 15% 3 .
however , pbc is suggested to only rarely be associated with rheumatoid arthritis ( ra ) , and the true prevalence of pbc in ra is not well known 4 . in this article
, we report an unusual case of a patient with pbc and ra , and discuss the association between these two diseases .
in 2013 , a 71yearold man with rheumatoid arthritis ( ra ) was admitted to our hospital with elevated transaminase levels .
he had been taking prednisone ( 6 mg / day ) and salazosulfapyridine ( 1000 mg / day ) for ra since 2012 ( fig .
1 ) . he reported no consumption of alcohol , recent travel , or sexual contact .
, he was icteric with mild pruritus . an abdominal ultrasound scan , computed tomography ( ct ) , and magnetic resonance cholangiopancreatography ( mrcp ) revealed neither biliary obstruction nor spaceoccupied lesions .
blood test results ( table 1 ) revealed the following : total bilirubin ( tbil ) , 3.76 mg / dl ; direct bilirubin ( dbil ) , 3.12 mg / dl ; aspartate aminotransferase ( ast ) , 167 iu / l ; alanine aminotransferase ( alt ) , 435 iu / l ; alkaline phosphatase ( alp ) , 2539 iu / l ; glutamyltranspeptidase ( gtp ) , 590 iu / l ; immunoglobulin g ( igg ) , 1322 mg / dl ; immunoglobulin m ( igm ) , 705 mg / dl ; antinuclear antibodies ( ana ) , 40 ; antimitochondrial antibodies ( ama ) , 20 ; amam2 antibodies , 34.1 ( normal ; < 6 ) ; antigp210 antibodies , 0.2 u / ml ( normal ; < 6 ) ; anticentromere antibodies , 55.6 u / ml ( normal ; < 9.9 ) . a liver biopsy at day 4 showed marked inflammatory cell infiltration surrounding and destroying the interlobular bile ducts in the portal area ( fig .
on the basis of these results , a diagnosis of pbc corresponding to sheuer 's stage i was confirmed .
treatment with ursodeoxycholic acid ( udca ) ( 600 mg / day ) was started 3 days after admission .
the patient 's clinical findings and biological data showed improvement and the patient was discharged 19 days after admission .
he has been followed up in our outpatient clinic . a second biopsy after 445 days of udca treatment showed marked improvement of inflammation in the portal area ( fig . 2b ) .
ast , aspartate aminotransferase ; alt , alanine aminotransferase ; alp , alkaline phosphatase ; gtp , glutamyltranspeptidase ; crp , creactive protein ; hbsag , hepatitis b surface antigen ; hcvab , hepatitis c virus antibody ; ebvvca , epstein barr virus viral capsid antigen ; ebna , epstein barr virus nuclear antigen ; ana , antinuclear antibodies ; asma , antismooth muscle antibodies ; ama , antimitochondrial antibodies ; antitg ab , antithyroglobulin antibody ; antitpo ab , anti thyroid peroxidase antibody .
( a ) a liver biopsy at day 4 . marked inflammatory cell infiltration is observed surrounding and destroying the interlobular bile ducts with ductular proliferation in the portal area .
hepatic involvement is common in rheumatic diseases and is usually related with nonspecific findings such as drugrelated liver dysfunction .
however , more serious hepatic involvement , including nodular regenerative hyperplasia , vasculitis , and pbc , have been observed in specific rheumatic diseases , such as ra 3 , 5 .
ra is present in about 1% of the population and pbc is even rarer , with an estimated prevalence of 0.02% in females and 0.002% in males .
however , there are some reports that describe their association with individuals or families 4 , 7 .
pbc patients often have concomitant autoimmune diseases , such as sjgren 's syndrome ( 70% ) , systemic sclerosis ( 15% ) , and thyroiditis ( 10% ) .
the association of pbc and ra is unclear , although previous studies on large series of pbc patients have suggested that the prevalence of ra in pbc is 1.85.6% 4 , 8 , 9 .
meanwhile , several previous studies have indicated the presence of pbcrelated features in ra patients .
one autopsy series of ra patients found that 65% of 182 ra cases had evident liver pathology , including chronic inflammatory infiltration of the portal tracts and small foci of necrosis , as well as steatosis 10 .
however , it is unclear whether any of these patients were diagnosed with pbc . a japanese study by takahashi et al .
furthermore , another study indicated ama positivity in ra patients to be 18% 12 . in our case
, ama testing was not performed at the diagnosis of ra because liver dysfunction was not observed . although the etiologic and pathologic mechanisms of these diseases are not yet understood , several factors have been suggested , such as genetic factors , epigenetic factors , and infectious agents .
genomewide association studies ( gwas ) have shed light on the genetic background of pbc and ra recently .
genetic studies have indicated that several genes implicated in pbc lay within nonhla and hla regions 13 , 14 .
several common genes have been identified in pbc and ra , although the majority of implicated genes in both diseases do not overlap .
overlapping genes include hladqb1 , ctla4 , mmel1 , irf5 , stat4 , and possibly cxcr5 . among these genes , stat4 is essential for il12 signal transduction via the il12 receptor ( il12r ) for ifn production and th1 polarization . in a gwas for pbc in the japanese population ,
15 reported that , in addition to possessing two significant susceptibility loci ( tnfsf15 and pou2af1 ) , stat4 showed a suggestive association with pbc in japanese and european patients . in our case
, we did not perform a genetic analysis ; however , individuals with a common genetic profile may be more susceptible to developing concomitant ra and pbc . assuming that several common genes exist in ra and pbc patients
, it has been suggested that common infectious triggers may be involved in the induction of both diseases .
most studied infection trigger of pbc is escherichia coli ( e. coli ) 16 ; due to the high incidence of recurrent urinary tract infections ( rutis ) in pbc patients 17 .
several infectious agents ( including e. coli ) have also been linked to ra 18 , 19 . of note , in rfpositive patients , antie .
no symptoms of bacterial infection , including rutis , were observed during the clinical course .
however , a possible role of e. coli has been suggested in the early pathogenesis of ra . in our case , the patients showed rapid elevation in alt as well as alp and gtp .
, druginduced liver injury ( dili ) and overlap syndrome between pbc and auto immune hepatitis ( aih ) were suspected at first .
however , the patient was finally diagnosed as pbc by laboratory data , histology findings , and clinical course . in our case ,
overlap syndrome between pbc and aih was also suspected , however , serum level of alt was normalized after 2 months of udca treatment .
for these findings , we thought that the diagnosis in our case was also closer to pbc than overlap syndrome . in conclusion ,
concomitant pbc in patients with ra is rare ; however , pbc should be ruled out in the differential diagnosis of any patients with ra having abnormal liver function tests . to determine the relationship between these two diseases ,
further studies are needed , in particular cohort studies related to pbc occurrence in a large cohort of ra patients .
| key clinical messagethe true prevalence of pbc in ra is not well known .
herein , we report an unusual case of a patient with pbc and ra , and discuss the association between these two diseases .
pbc should be ruled out in the differential diagnosis of patients with ra having abnormal liver function tests . |
it has been recently substantiated the psychological impacts of acne appearance and proved its - related negative emotional reactions in the patients
. the emotional impact of acne can be difficultly predictable because of the presence of many underlying factors is varied in different populations and thus identifying these determinants seems to be necessary .
acne is a chronic inflammatory disorder of the skin that most commonly presents during adolescence , a period with psychological instability .
it has been known as a ubiquitous affliction with physical and emotional scars that persists throughout the life of the affected individuals . besides experience of impaired functional status and decreased quality of life , it has been recently substantiated the psychological impacts of acne appearance and proved its - related negative emotional reactions in the patients .
for instance , the occurrence of acne substantially augments the enormous stress that burdens most women , particularly the younger in today 's demanding society .
depression and anxiety have been suggested more prevalent among patients with acne than among control subjects.[36 ] the results of some studies indicated a higher level of emotional and social impairment , in terms of the feelings of physical discomfort , anger , and the intermingling impact of these , among these patients .
moreover , higher overall psychiatric morbidity in those with severe acne compared to normal population has been revealed.[810 ] however , the emotional impact of acne can be difficultly predictable because of the presence of many underlying factors such as patients age and gender , psychosocial developmental period , clinical severity of the disease , family and peer support systems , personality coping styles , and other underlying psychopathology .
thus , the impact of acne appearance on psychological status of individuals might be varied in different populations .
the current study investigates whether acne and its severity affects psychological functioning in those who suffered from this disorder among iranians .
one hundred and six patients with acne vulgaris who consecutively attended the dermatology outpatient clinics in semnan city in 2008 were included . among them , 103 patients met the study 's inclusion criterion and agreed to participate .
the study participants or their family members or relatives had no history of other skin disorders as well as no history of psychological problems .
also , all participants were in intermediate economic level [ table 1 ] . moreover , none of them tended to special dietary patterns .
the participants also had no history of anti - psychotic or anti - depressive drugs .
because of the potential effects of acne complications on late psychological events , the patients with these complications such as acne - related scars were not included into the study .
one hundred and six age and gender cross - matched healthy volunteers were included as controls that attended the clinic with their diseased relatives .
the institutional ethical committee approved the study design and informed consent was obtained from all patients and controls .
sociodemographic and clinical characteristics of the patients and the controls the demographic and clinical questionnaires collected data on the demographic characteristics of the patients ( age and gender ) , acne ( localization , duration , and severity ) and presence or absence of daily activity disturbance that considered as a criterion for need to treatment .
acne severity was classified as mild , moderate , or severe according to the classification of the american academy of dermatology as : ( 1 ) mild acne : characterized by the presence of a few papules and pustules mixed with comedones , but no nodules ; ( 2 ) moderate acne : characterized by the presence of many papules and pustules , together with a few nodules , and ( 3 ) severe acne : characterized by the presence of numerous or extensive papules and pustules , as well as many nodules .
also , acne in participants was studied regarding its distribution and evaluated in 4 classes : mostly the face and neck , mostly the body , mostly the limbs , and equally generally distributed .
all acne patients were evaluated using the symptom check list-90 ( scl-90 ) that was firstly developed by derogatis et al . , and was translated into persian and its acceptable reliability and validity was proved among iranian population .
each item of the 90 is rated on a five - point scale of distress ( 0 - 4 ) , ranging from not at all to extremely .
psychological symptoms include somatization , obsessive compulsive , interpersonal sensitivity , depression , anxiety , hostility , phobic anxiety , paranoid ideation , and psychoticism . under usual circumstances
we proposed that the cut - off point equaled the sum of one mean + standard deviation , which also equaled a t - score of 60 for each item .
patients with a score equal to or greater than the cut - off point are in need of detailed psychiatric assessment and consultant ; as such scores indicate the presence of clinically significant symptoms .
for statistically analysis , comparison of categorical variables between the independent groups was performed using chi - square test and for determining the relationship between constant variables , pearson 's correlation coefficient was used .
all the statistical analyses were performed using spss version 15.0 ( spss inc . , chicago , il , usa ) .
the patient group ( 103 ) consisted of 71 ( 68.9% ) women and 32 ( 31.1% ) men . of the 106 controls , 60 ( 56.6% ) were women and 46 ( 43.4% ) were men .
the mean ages of the patients and controls were 26.0 8.7 and 26.4 8.2 years , respectively .
there was no significant difference between patients and controls with regard to age , gender , marital status , education level , as well as economic condition [ table 1 ] . according to the american academy of dermatology classification , 25.2% of the patients had mild acne , 50.5% moderate acne , and 24.3% severe acne .
with regard to the duration of disease , 31.1% of patients had duration of less than 2 years , 33.0% had duration of 2 to 5 years and 35.9% had duration of 5 years or more . in almost 72.8% of the patients ,
acne localization was on the face and the neck , in 14.6% of them acne was mostly localized on the body , in 1.0% of the patients , acne was localized on the limbs and in others acne was generally distributed . as shown in table 2 , a higher percentage of participants than controls required further evaluation and psychological consultant when studying obsession ( 50.5 , p < 0.001 ) , sensitivity ( 46.7% , p < 0.001 ) , phobia ( 46.6% , p < 0.001 ) , hostility ( 46.5% , p < 0.001 ) , anxiety ( 41.8% , p < 0.001 ) , paranoid ideation ( 34.2% , p
< 0.001 ) , somatization ( 34.0% , p < 0.001 ) , depression ( 22.3% , p = 0.001 ) , and psychoticism ( 17.3% , p = 0.009 ) due to the appearance of primary psychological manifestations . relative frequency of patients and controls that require further evaluation and psychological consultant according to their symptom check list-90 scores the most common psychological symptoms requiring treatment because of disturbed daily activities in acne group were psychoticism ( 34.0% ) and depression ( 31.1% ) , respectively .
also , a higher percentage of participants than controls required beginning of the treatment when studying these two psychological symptoms as well as hostility and interpersonal sensitivity [ table 3 ] . relative frequency of patients and controls that require psychological treatment according to their symptom check list-90 scores statistically significant positive correlations were observed between the duration of illness and scl-90 total score ( r = 0.456 , p = 0.002 ) . when evaluating the scl-90 scores , patients with multiple sites of involvement
were affected more severely than those with a single site of involvement ( p = 0.026 ) .
the results of the current study revealed the negative influence of acne vulgaris on patients psychological status that all psychiatric symptoms such as somatization , obsession , sensitivity , depression , anxiety , hostility , phobia , paranoid ideation , and psychoticism were associated with this skin disorder . on the other hand , participants with acne had higher scores than controls in all scl-90 items .
furthermore , the more severe acne and the longer its duration resulted in the worse psychological manifestations . our findings could support those of many researches that have addressed the relationship between acne and psychological symptoms . in a study by abdel - hafez study ,
acne patients had significantly higher scores than controls in all items of the scl-90 . in a qualitative study by magin et al .
, psychological morbidity was considerable in acne patients and more prominent symptoms were embarrassment , impaired self - image , low self - esteem , self - consciousness , frustration , and anger .
furthermore , in a similar study on iranian patients , prevalence of anxiety and mean of anxiety scores were 68.3% and 9.17 3.52 , respectively , in patients group and 39.1% and 7.10 3.07 , respectively , in control group in which there was a significant difference .
it seems that high failure to seek appropriate medical treatments can results in disappointment of acne sufferers and therefore these patients might experience the initial symptoms of psychological problems .
it was shown that out of approximately 2.7 to 3.5 million acne sufferers , only some 500,000 are presently undergoing medical treatment .
failure to seek medical treatment is closely associated with the fact that acne sufferers for the most part expect to be disappointed with the results .
our study also showed that although most of the study participants needed to psychological consultant because of their initial psychological symptoms , only overall incidence of depression and psychoticism were considerably high in the patients .
we showed that the prevalence of these two problems was 31.1% and 34.0% , respectively , while the prevalence of other psychological aspects was lower than 4.0% .
in some recent studies , 8% to 26% of patients with acne have been reported to suffer from at least mild depressive symptoms.[1720 ] however , in some others , the rates of depressive symptoms in acne patients were considerably low that the acne did not seem to be related to depressive symptoms . based on our findings , the rates of depressive disorders and psychoticism can be high in acne patients in our population and therefore psychological consultations should be done in all acne patients , particularly in those with primary symptoms and primary diagnosis of these two problems . besides , effective treatment of acne can be accompanied by improvement in psychological impairment,[2325 ] and therefore it seems that the patients with acne and concurrent appearance of the symptoms of psychological impairment require more appropriate acne therapy . in this
regard , psychiatric consultation should be sought by dermatologists and patients should be followed with the cooperation of dermatologists and psychiatrists .
totally , effective concomitant anti - acne therapy and psychological assessment make significant contributions for the mental health and should be strongly recommended .
to confirm different aspects of psychological disorders in patient with acne , we evaluated these aspects and showed that effective concomitant anti - acne therapy and psychological assessment in these patients are necessary . | background : the psychological impacts of acne appearance and its - related negative emotional reactions have been proved ; however , these reactions are varied in different populations.aim:we investigated whether acne and its severity affected psychological functioning in those who suffered from this disorder among iranians.materials and methods : one hundred and six patients with acne vulgaris who consecutively attended the dermatology outpatient clinics in semnan city in 2008 were included . among them , 103 patients met the study 's inclusion criterion and agreed to participate .
one hundred and six age and gender cross - matched healthy volunteers were included as controls that attended the clinic with their diseased relatives .
all acne patients were evaluated using the symptom check list-90 ( scl-90).results : according to the american academy of dermatology classification , 25.2% of the patients had mild acne , 50.5% moderate acne , and 24.3% severe acne . a higher percentage of participants than controls required further evaluation and psychological consultant when studying each psychological problem .
the most common psychological symptoms requiring treatment due to disturbed daily activities in acne group were psychoticism ( 34.0% ) and depression ( 31.1% ) , respectively .
significant positive correlations were observed between the duration of illness and scl-90 total score .
when evaluating the scl-90 scores , patients with multiple sites of involvement were affected more severely than those with a single site of involvement.conclusion:acne vulgaris has significant effects on psychological status .
effective concomitant anti - acne therapy and psychological assessment make significant contributions for the mental health and should be strongly recommended . |
adrenal myelolipomas are benign tumors which are usually asymptomatic and diagnosed incidentally on imaging or during autopsy .
they were first described by gierke in 1905 and were termed as myelolipomas by oberling in 1929 [ 1 , 2 ] .
they are uncommon benign tumors of the adrenal cortex constituting approximately for 35% of all primary adrenal tumors and are increasingly being diagnosed due to improved imaging techniques [ 3 , 4 ] .
adrenal myelolipomas are most frequently detected after the fifth decade and are seen almost equally in both genders .
they are commonly found as unilateral masses , but occasional bilateral masses have been documented .
they are usually asymptomatic but myelolipomas may attain large sizes and present as abdominal masses [ 7 , 8 ] .
diagnosis of giant adrenal myelolipomas in a timely manner may prevent complications due to hemorrhage and can be lifesaving .
a 52-year - old man with a past medical history significant for atrial fibrillation , diabetes type 2 and hypertension for 30 years was admitted with early satiety for the past 3 months and progressive dyspnea on exertion for 3 weeks .
he also complained of gradual unintentional weight loss of approximately 100 pounds over the last 10 years .
he had been a non - smoker and a moderate alcohol user for the last 30 years . on initial presentation to the hospital
, he denied shortness of breath at rest or chest discomfort and was in no acute distress .
his blood pressure was 204/93 mm hg on the left arm and 194/88 mm hg on the right arm with an irregular heart rate of 54 beats / min and oxygen saturation of 96% on room air .
the lungs were clear to auscultation , jugular venous distension was noted at 13 cm above the sternal angle and 3 + bilateral leg edema with scrotal edema .
his abdomen felt firm and lacking in tympanicity , but it was non - tender in all four quadrants .
laboratory values were notable for a brain natriuretic peptide of 980 ng / l , creatinine of 1.2 mg / dl , bun of 20 mg / dl , hco3 of 31 meq / l , inr of 1.23 , hb of 12.5 g / dl , na of 140 meq / l , k of 3.7
he was monitored in the intensive care unit for decompensated heart failure with uncontrolled hypertension . to promote diuresis and reduce afterload ,
he was administered intravenous furosemide 80 mg , oral metolazone 2.5 mg , oral hydralazine 75 mg every 6 h , oral clonidine 0.3 mg , lisinopril 40 mg , isosorbide mononitrate 120 mg and losartan 100 mg .
overnight , he had an episode of moderate self - limited epistaxis with minimal improvement in blood pressure control .
he remained in slow atrial fibrillation with a lowest recorded heart rate of 38 beats / min .
however , he denied dizziness at any time despite a negative fluid balance of 1.8 liters .
an echocardiogram showed a left ventricular ejection fraction of 30% with four - chamber dilatation , moderately reduced right ventricular systolic function , severe mitral regurgitation and moderate pulmonary hypertension .
he underwent right heart catheterization showing a right atrial pressure of 12 mm hg , right ventricular pressure ( systolic / diastolic ) of 60/12 mm hg , pulmonary artery pressure of 60/23 mm hg and pulmonary capillary wedge pressure of 20 mm hg and a cardiac index of 2.3 liters / min .
a left heart catheterization revealed clean coronaries , and alcohol was thought to be the likely etiology of non - ischemic cardiomyopathy in this patient .
urine catecholamine and vanillylmandelic acid were measured and a magnetic resonance angiography of the renal arteries was performed to evaluate for secondary causes of hypertension .
this showed non - obstructed renal arteries bilaterally . a minimally enhancing 22 16 23
cm mass was visualized in the left upper and mid - abdomen displacing the spleen and stomach posterosuperiorly , the pancreas anteriorly and the left kidney inferiorly ( fig .
a contrast - enhanced ct scan of the abdomen to further characterize the mass showed no infiltration of the surrounding vasculature or organs , and the left adrenal gland was visualized although it could not be confirmed if the mass was arising from the adrenals ( fig .
a needle biopsy of this mass showed adipose tissue with foci of extramedullary hematopoiesis consistent with myelolipoma without any evidence of malignancy , confirming this mass to be a benign myelolipoma ( fig .
a renal perfusion scan showed a small left kidney with reduced perfusion contributing 22% to the overall tubular function without obstructive uropathy .
the lower extremity edema was partly ascribed to be due to inferior vena cava compression , and diuretics were decreased due to worsening renal function . given the bradycardia and severe congestive heart failure , an electrophysiologist was consulted for evaluation and need for pacemaker implantation .
bradycardia was ascribed to be partly due to the cushing reflex secondary to elevated blood pressure , and as the patient had no symptoms secondary to a low heart rate , pacemaker implantation was not advised . despite maximum doses of clonidine , isosorbide , hydralazine , losartan and lisinopril , at
, the mass was found to be soft , highly vascular but well encapsulated and attached only to the left adrenal gland via a small portion that was resected along with the mass ( fig .
there was minimal blood loss during the surgery , and the patient had an uneventful recovery in the postoperative period . a renal perfusion scan done 8 days after surgical removal showed 36% contribution to the overall tubular function .
blood pressure control improved after surgery and the patient was gradually titrated off clonidine , hydralazine and lisinopril .
bradycardia resolved with few episodes of atrial fibrillation with rapid ventricular response that were controlled with -blockers and digoxin .
the antihypertensive medications that the patient was discharged on were carvedilol 40 mg , valsartan 320 mg and isosorbide mononitrate 240 mg .
they are thought to arise from the reticuloendothelial cells of the adrenal gland in response to repeated stimulation by stress , inflammation and acth [ 11 , 12 , 13 ] .
both the adipose and the hematopoietic cells have been noted to have the same pattern of x - chromosome inactivation and have been postulated to arise by clonal proliferation from a common mesenchymal stem cell . moreover ,
the chromosomal translocation ( 3;21)(q25;p11 ) has been detected in myelolipomas in patients with hematological malignancies , which has led to the speculation of bone marrow origin of these tumors .
extra - adrenal myelolipomas have been documented in the mediastinum , lung , thoracic spine , liver mesentery , spleen , kidney and pelvis [ 16 , 17 , 18 , 19 , 20 , 21 ] .
extra - adrenal myelolipomas may mimic extramedullary hematopoietic tumors especially since bone spicules have been reported .
extramedullary hematopoietic tumors generally have a higher content of erythroid cells and less fat tissue compared to extra - adrenal myelolipomas which have a relatively higher number of lymphoid cells .
however , calcification is seen in approximately 27% of the cases and the presence of a higher proportion of hematopoietic tissues may mimic an adrenal adenoma [ 4 , 25 , 26 ] .
ultrasonography is not a very useful diagnostic modality , especially if the tumors are smaller in size ( < 2 cm ) .
an mri has a similar diagnostic utility as a ct scan in adrenal myelolipomas with a high fat content .
the gold standard for diagnosis is tissue biopsy which typically shows hematopoietic cells admixed with mature adipose cells .
adrenal myelolipomas can be associated with hyperaldosteronism , congenital adrenal hyperplasia and cushing 's syndrome .
they are known to be biochemically inactive but a case of adrenal myelolipoma secreting cortisol has been reported .
adrenal myelolipomas < 5 cm are conservatively managed with sequential imaging , but larger masses are preferably resected due to the risk of hemorrhage .
laparoscopic resection has been successfully used to reduce hospital stay and improve perioperative outcomes , but it is not preferable for tumors > 10 cm or in tumors with infiltration or extensive adhesions [ 30 , 31 ] .
his clinical presentation was compatible with symptoms related to mass effect of the giant myelolipoma , which resolved after surgical resection .
their insidious clinical course becomes apparent only when mass effects are manifested in the form of an abdominal mass , flank pain , hypertension and pedal edema .
it is important to differentiate them from other malignancies , such as liposarcoma , and to ensure that the myelolipoma is not biochemically active .
adequate follow - up of small masses and surgical resection of large masses are the standard of care which provide for lesser morbidity and mortality .
| adrenal myelolipomas are rare benign tumors of the adrenal cortex composed of adipose and hematopoietic cells .
they have been postulated to arise from repeated stimulation by stress , inflammation and acth oversecretion .
myelolipomas are usually detected incidentally on imaging and do not require any active intervention besides regular follow - up by imaging .
however , myelolipomas may insidiously grow to large sizes and cause mass effects and hemorrhage .
timely diagnosis and surgical resection are curative and lifesaving . |
idiopathic orbital pseudotumor ( iop ) , also known as idiopathic orbital inflammatory syndrome ( iois ) , is a nonmalignant orbital inflammation without a known local or systemic cause .
it is the most common cause of a painful orbital mass in adults after graves disease .
diagnosis is one of exclusion based on history of the case , clinical examination and clinical course , imaging findings , biopsy , and response to steroid therapy .
iop can affect people of virtually any age , with no sex and race predilection .
the disease may be confined to a single orbital structure , but frequently may involve a combination of different structures including the globe , extraocular muscles , retroocular fat tissue , and lachrymal glands .
. clinical presentation may vary according to the specific location and degree of inflammation , fibrosis , and mass effect .
patients may present with proptosis , lid swelling , chemosis , limited motility with tenderness to palpation and pain on movement of the eyes , double vision , light sensitivity , and less commonly , vision loss .
an unusual case of iop with bilateral massive orbital infiltration extending into maxillofacial regions is presented in this report and its distinctive magnetic resonance imaging ( mri ) features that help to exclude other differential diagnoses are discussed .
a 25-year - old woman presented to our ophthalmology clinic with a long history of progressive protrusion of bilateral eyes and periorbital swelling without vision changes .
her symptoms started when she was 9 years old , but she had no history of treatment or operation because of poor conditions of her country .
clinical examination revealed protrusion of both eyes with bilateral conjunctival congestion , restrictive eye movements , and periorbital and facial swelling , more so on the right .
routine laboratory and hematologic investigations including t3 , t4 , and thyroid stimulating hormone ( tsh ) were normal .
doppler ultrasound ( usg ) showed increased soft - tissue thickness in the periorbital region with arterial and venous flow inside .
a vascular malformation was suspected and patient underwent routine and contrast - enhanced orbital mri [ figure 1 ] .
edematous contrast - enhancing soft - tissue thickening was noted in both orbits as well as in buccal and zygomatic regions . in the orbits , pre- and post - septal structures
were extensively affected , sparing the optic nerve dural sheaths and bulbi oculi on both sides .
vascular malformation graves disease was suspected . however , hypointensity seen on t2 image indicated infiltrative changes and ruled out graves disease .
extensions into paranasal sinuses , middle cranial fossa , and cavernous sinuses were not present .
massive orbital and maxillofacial infiltrating disease with t2 hypointensity , diffusion restriction , and multispatial appearance were suggestive of either lymphoproliferative diseases ( orbital pseudotumor , eosinophilic tumor , histiocytic tumor , langerhans cell tumor , periorbital xantogranulomatous diseases ) or sarcoidosis .
patient was treated with oral corticosteroids in tapering dose for 3 months . at the end of treatment ,
25-year - old woman with a long history of progressive bilateral protrusion of eyes , periorbital swelling without vision changes presented with intermittent attacks of retrobulbar pain and headache later diagnosed with idiopathic orbital pseudotumor .
( a ) axial t2 , ( b ) axial fat - saturated t1 , ( c ) axial post - contrast fat - saturated t1 , ( d ) coronal fat - saturated t2 , and ( e ) coronal fat - saturated t1-weighted images with ( f and g ) diffusion - weighted images with corresponding ( h and i ) apparent diffusion coefficient ( adc ) maps demonstrate multispatial massive infiltrative changes including pre - septal ( long thick arrows ) and post - septal orbital structures ( thin arrows ) and soft tissue of buccal ( asterisks on d and e ) and zygomatic regions ( arrowheads on c - e ) . infiltrated areas are enhancing , t2 hypointense in appearance with restricted diffusion ( arrows on f and i ) , suggestive of either lymphoproliferative diseases or sarcoidosis .
iop is a disease of unknown etiology and is characterized by non - granulomatous inflammation of the orbital soft tissue .
the inflammatory process is generally unilateral and may involve the extraocular muscles , orbital fat , sclera , optic nerve , or lacrimal gland .
iop can extend via the superior orbital fissure and optic canal into the middle cranial fossa and cavernous sinus , and via the inferior orbital fissure into the infratemporal and pterygopalatine fossa .
the most common imaging findings in decreasing order of frequency include infiltration of retroorbital fat , proptosis , extraocular muscle enlargement and enhancement , orbital apex inflammation , and optic nerve thickening .
mri is more valuable in demonstrating soft - tissue changes of iop because the fibrous and hypercellular nature of the inflammatory mass is reflected by its low signal intensity on t1-weighted ( t1w ) and t2-weighted ( t2w ) images .
orbital and systemic diseases that mimic iop include orbital cellulitis , graves disease , sarcoidosis , lymphoma , vascular malformation , metastatic carcinoma , uveal melanoma , and ruptured dermoid cyst .
these often can be distinguished based on radiographic characteristics on computed tomography ( ct ) and mri , along with clinical examination . in case of extensive orbital infiltration , orbital cellulitis , lymphoproliferative diseases ( iop , eosinophilic tumor , histiocytic tumor , langerhans cell tumor , periorbital xantogranulomatous diseases ) , graves disease , and sarcoidosis , vascular malformation should be considered in differential diagnosis .
orbital cellulitis is an infective process associated with pain , fever , and sinusitis , and is usually unilateral .
multispatial appearance favors both vascular malformation and lymphoproliferative diseases , but t2 hyperintensity is typical for vascular malformation .
iop can mimic thyroid opthalmopathy ; but in iop , the involvement is less regular and unilateral with involvement of the tendinous insertion of the extraocular muscles .
infiltrative changes in the adjacent fat , periocular involvement , or a discrete mass with thickened muscles favors the diagnosis of iop . in graves disease , the affected extraocular muscles are t2 hyperintense in contrast to the findings in lymphoproliferative disease . in severe cases of graves disease ,
ophthalmologic involvement is frequent , and anterior uveitis , retrobulbar masses , or infiltration of lacrimal gland , extraocular muscles and optic nerve , chiasma , and/or sheath , eyelid swelling , palpable lid masses , and bone destruction can be seen .
infiltrations in sarcoidosis are similar to those of lymphoproliferative diseases with the lesion being isointense on t1w and hypointense on t2w images , but no diffusion restriction is reported in literature .
our case is unusual in that the clinical course was very long in duration , presentation was bilateral , and there was associated massive involvement of the subcutaneous tissues of the buccal and zygomatic regions on both sides .
however , imaging features showing multispatial appearance , t2 hypointensity , and diffusion restriction greatly decreased the possible entities in differential diagnosis .
diagnosis of iop is one of exclusion because many other conditions may mimic this lesion ; thus , it represents a diagnostic challenge for radiologists and clinicians .
complete and detailed medical history , relevant serologic tests , and imaging studies are important to exclude other conditions .
iop should be considered in patients with clinical evidence of persistent , progressive , or recurrent orbital disease and mri features show multispatial involvement , t2 hypointensity , and diff usion restriction . | idiopathic orbital pseudotumor ( iop ) is a benign inflammatory condition of the orbit without identifiable local or systemic causes .
bilateral massive orbital involvement and extraorbital extension of the iop is very rare .
we present an unusual case of iop with bilateral massive orbital infiltration extending into maxillofacial regions and discuss its distinctive magnetic resonance imaging ( mri ) features that help to exclude other entities during differential diagnoses . |
repetitive thoughts have been defined as the process of thinking attentively , repetitively or frequently about one 's self and one 's world [ 1 , page 909 ] .
their constructive properties are discussed in terms of enhanced adaptive preparation , anticipatory planning , and others ( for an overview see ) . amongst repetitive thoughts ,
those of a ruminating style characterized by depressive contents ( depressive rumination ) are seen as particularly unconstructive and maladaptive .
depressive rumination has been defined in various ways ( see ) , with one of the most frequently used provided by nolen - hoeksema and colleagues who conceptualize depressive rumination as behaviors and thoughts that focus one 's attention on one 's depressive symptoms and on the implications of these symptoms [ 4 , page 569 ] .
using this definition , the current paper investigates depressive rumination as a risk factor for depression and a personality trait that differentiates between healthy individuals and their individual levels of nonclinical depression .
the concept of depressive rumination was introduced several years ago within the framework of nolen - hoeksema 's response styles theory ( rst , [ 57 ] ) .
ruminators tend to remain fixated on the problems and on their feelings about them without taking action .
previous research suggests that depressive rumination intensifies depressed mood and predicts the onset , recurrence , severity , and duration of depressive episodes [ 8 , 9 ] .
ruminators are ineffective in active , interpersonal problem solving [ 10 , 11 ] , and a depressive ruminative response style leads to more pessimistic future perspectives and less social support [ 12 , 13 ] .
for example , in a recent study , rumination was shown to foster indecision , indicating that rumination interferes with decision making in dysphoric individuals .
in addition , depressive rumination has been demonstrated to mediate several other hypothesized risk factors that prospectively predict the number of depressive episodes , including dysfunctional attitudes , neediness , self - criticism , and history of past depression . in a sample of patients with fatal neurodegenerative disease ,
depressive rumination was found to mediate the negative effect of reminiscence on well - being .
this suggests that a ruminative response style is an important factor for both the onset and maintenance of depression , and that it is an important target for treatments aimed at reducing current and future affective symptoms .
previous research on ruminative thinking has mainly focused on affective components of well - being , such as depression . beyond affect ,
a distinct cognitive component of subjective well - being has been referred to as life satisfaction .
life satisfaction refers to a global , cognitive - judgmental aspect of a person 's life , based on one 's own standards , goals , and weightings of various life domains , and plays a complementary role for the judgment of subjective well - being [ 18 , 19 ] .
life satisfaction is negatively correlated with depression [ 20 , 21 ] but is more sensitive to change and shows some degree of independence from purely affective constructs , including depression . given that both depression and life satisfaction are known to change over the lifespan and are associated with depressive rumination , this triangular relationship needs to be investigated with regard to age - related effects . to our knowledge
, the relationship between ruminative response styles and global life satisfaction has not yet been investigated with respect to the influence of aging . considering the partial independence of the purely affective construct of depression and the more cognitive - judgmental construct of life satisfaction , the current study aimed at investigating specific influences of ruminative styles on both measures of subjective well - being .
studies investigating rumination on the background of the rst have typically used the 22-item rumination reponse scale ( rrs ) of the response style questionnaire ( rsq , ) .
their results support a two - factor model of rumination , which includes the components of reflective pondering and brooding , the latter of which was found to be the key factor in the prediction of depressive symptoms .
relates to passive and self - critical thoughts comparing one 's current situation against a desired standard or goal [ 23 , page 605 ] , whereas reflection refers to a more purposeful inward examination and attempt at problem - solving in response to depressed mood
armey and colleagues replicated these results such that they identified the same two - factor structure as treynor and colleagues with brooding being more strongly related to depression than pondering . taken together these results provide accumulating evidence that rumination is a key factor in depression .
nevertheless , studies investigating the association between rumination , depression , and life satisfaction over the lifespan are rare .
the majority of studies , which address the two postulated components of rumination , either examined primarily undergraduate students or did not address age as a variable under investigation , although clinical and epidemiologic findings suggest that depression and life satisfaction are age dependent .
adolescence , for example , has been repeatedly identified as a critical period in the lifespan for the onset of a range of mental disorders , including depression and anxiety .
the majority of results show a peak in adolescence and a subsequent decline in incidence and prevalence for most mental disorders with increasing age [ 2527 ] .
the reasons for these associations have been discussed in terms of changing life circumstances , critical life events , and other external factors determining the presence of stressors , as well as neurodevelopmental causes [ 28 , 29 ] .
other factors for these age - dependent effects may be explained by findings indicating decreased emotional responsiveness with age , increased emotional control , and psychological immunization to stressful experience .
based on these results , we hypothesize that rumination , which is known to be associated with the occurrence of depression , to be more pronounced in early adulthood , and to be lowest in older adults . at the other end of the lifespan and supporting the hypothesis of an inverse association between aging and rumination , studies examining age differences in emotional experiences have dispelled the myth of age - related decline of well - being . for decades , this decline was taken as common sense , probably caused by assumptions based on the decline in physiological functions and the increase in negative life events such as loss of friends .
older age is not associated with increased emotional distress ; to the contrary , there is even a slight decrease in self - reported negative affect in older adults compared to middle - aged and younger adults , as well as lower rates of anxiety and major depression [ 33 , 34 ] .
a steady decline in subclinical depression has been reported across young , middle and older adulthood . in terms of lifetime prevalence , 1% of older adults
are diagnosed with major depression , compared to 6% of younger adults . with the exception of a slight age - related increase in depressive symptoms reported in some studies , the majority of studies found depression levels to be lower in older compared to younger adults [ 31 , 33 ] .
it has to be conceded , however , that a decreasing prevalence of major depression with age is not equivalent to decreased depression levels per se , as minor , subsyndromal depression is usually not caught by studies focusing on major depression
. the current study will thus discuss depression scores obtained using a self - report questionnaire designed for nonclinical populations .
a better understanding of the age - dependent associations of reflecting , brooding , depression and life satisfaction in a nonclinical sample may help in the prevention of mental ill - health and contribute to the development of cohort - specific therapeutic interventions . in the current study , first , a replication of treynor and armey 's [ 22 , 24 ] two - factor structure was conducted to ensure the implementation of identical factors as described in previous research .
this attempted replication was based on the response style questionnaire 's ( rsq ) 10-item rumination subscale , as extracted from treynor and colleagues .
second , we hypothesized that depressive rumination would be most pronounced at a younger age and would be lowest in the oldest investigated age group .
third , we explored the relative contribution of the different ruminative styles of brooding and reflection to affective ( i.e. , depression ) and cognitive - judgmental aspects ( i.e. , life satisfaction ) of subjective well - being .
they were approached in public settings ( e.g. , cafes , retirement communities , long distance trains , public squares ) using ad hoc recruitment .
the remaining 299 participants ( 118 women ) were aged between 15 and 87 years ( m = 41.90 , sd = 18.57 ) .
a total of 269 ( 89.7% ) participants were native german speakers , the remaining 31 ( 10.3% ) participants spoke fluently german .
one hundred and eleven ( 37.0% ) participants were single , 76 ( 25.3% ) were living in a partnership , 78 ( 26% ) were married , 22 ( 7.3% ) were divorced , and 13 ( 4.3% ) widowed .
a total of 89 persons ( 30.1% ) completed secondary school , 173 participants ( 58.4% ) achieved a university entrance qualification .
assignment to age categories followed the world health organization 's definition of youth encompassing the age range of 1524 .
seniors were defined as 63 years of age or older , following the actual pensionable age in germany according to the national statistics office ( http://www.destatis.de/ ) .
participants with an age between 24 and 63 years were divided into three groups with approximately equal age ranges of 12 and 11 years .
table 1 shows proportions of participants in each age category together with their mean age and standard deviation .
rumination style was assessed with treynor and colleagues ' version of the response style questionnaire ( rsq ) , which assesses ruminative styles on the subscales brooding ( cronbach 's alpha = .72 ) and reflective pondering ( cronbach 's alpha = .60 ) .
both subscales consist of five items to be answered on a 4-point likert - scale ( e.g. , brooding : what am i doing to deserve this ? or reflective pondering :
i analyze recent events and try to understand why i am depressed . ) .
the german translation applied in this study reached comparable internal consistency ( cronbach 's alpha = .69 for subscale brooding ; cronbach 's alpha = .75 for subscale reflective pondering ) .
depression was assessed with the allgemeine depressivitts - skala langversion ( ads - l , ) , which is the german version of the center of epidemiologic studies depression scale ( ces - d ; ) .
the ces - d was developed to assess depressive symptoms in nonclinical populations . with its high sensitivity ,
the questionnaire is suitable to assess interindividual differences in depression in highly functional , nonclinical samples .
a total score is calculated from 20 items to be rated on a 4-point likert - scale ( e.g. ,
the ces - d focuses on current states . in representative nonclinical samples , split - half reliability ( r = .89 ) and test - retest reliability ( r = .45.70 , depending on the time interval )
satisfaction with life was assessed using the satisfaction with life scale ( swls ; ) .
the swls assesses cognitive - judgmental aspects of subjective well - being based on a standard that each individual sets for him or herself .
five items are responded to on a 6-point likert - scale ; scores can range from 5 ( low satisfaction ) to 30 ( high satisfaction ) points ( e.g. , in most ways my life is close to my ideal ,
if i could live my life over , i would change almost nothing ) .
the two - month test - retest correlation coefficient was .82 , and cronbach 's alpha was .87 .
a factor replication was carried out to validate the german translation of the rsq and to ensure that the factor structure reported in the original version is independent of age . to investigate the dimensionality of the rumination scale , mokken scale analysis ( msa ) was used .
mokken scale analysis is a form of nonparametric item response theory ( nirt ) , which consists of a family of measurement models that are based on a minimal set of assumptions ; making them especially appropriate for the scale analysis of ordinal data such as rating scales [ 4043 ] .
mokken scale analysis was applied using the software package mokken scale analysis for polytomous items ( msp5.0 , ) .
we used the double monotonicity model ( dmm ) , which is based on four assumptions : unidimensionality , local independence , monotonicity , and double monotonicity .
unidimensionality : the items measure one latent trait only ( referred to as ) .
local independence : the scale consists of items which the participant approaches in a way that is independent of the previous items .
monotonicity : the item response functions ( irfs ) should be monotone nondecreasing ( monotonicity ) .
this means that an increase in -level never corresponds with a decrease in the probability of choosing item category m or higher .
together , these three assumptions result in a measurement model which can be used to rank - order respondents on an underlying unidimensional scale using the unweighted sum of item scores [ 42 , 4547 ] .
it is assumed that the i(s)rfs ( see endnote ) do not intersect across the latent trait .
this assumption holds if there is an unambiguous rank ordering of items and response categories within each item .
if this assumption holds , the items can be unambiguously ordered on the underlying trait [ 42 , 43 ] . in order to evaluate whether the scale or scales are unidimensional ,
these coefficients are calculated between item pairs ( hij ) , on the item level ( hi ) , and on the scale level ( h ) .
hij equals the items ' covariance corrected for the maximum covariance given the items ' univariate score - frequency distributions .
an important advantage of this statistic is that it avoids problems with respect to the distorting effect of difference in item - score distributions on inter item correlations .
the his are based on the hijs and express the degree to which an item is related to other items in the scale .
h is based on the his and expresses the degree to which the total score accurately orders persons on the latent trait scale .
a scale is considered acceptable if .3 h < 0.4 , good if .4 h < .5 , and strong if h .5 [ 42 , 49 ] .
we tested two scale solutions : one assuming a unidimensional structure ( rumination ) , and one assuming a two - dimensional structure ( brooding and reflection ) .
in the first analysis , all rsq items were entered . in the second analysis ,
these two analyses were then compared and the best solution was chosen based on the h - values and the aforementioned assumptions .
a one - way between groups multivariate analysis of variance was performed to investigate group effects of age on ruminative style .
the two dependent variables were the subscale brooding of the rsq and subscale reflective pondering of the rsq .
preliminary testing checked for normality , linearity , univariate and multivariate outliers , homogeneity of variance - covariance matrices , and multicollinearity , with no serious violations noted . in a second step , a regression analysis was used to model the relationship between the independent variables reflective pondering / brooding and the dependent variables depression / life satisfaction , while controlling for sex and age and investigating all interaction effects between sex , age , and the two rumination subscales .
separate regression analyses were used for depression and life satisfaction . for factors sex and age , the reference categories used were male and age between 2537 years , respectively . to test whether the relationship between the independent variables and dependent variables was dependent on sex or age group ,
the models were built using a forward procedure , adding one variable at a time .
we started with the control variables sex and age and continued by adding brooding , reflective pondering , and the interaction effects .
self - report measures were within normal range ( table 2 ) compared with previous reports [ 18 , 22 , 38 ] .
all items were included in one scale , whereas analysis 2 tested the two hypothesized subscales of rumination ( i.e. , brooding and reflective pondering ) . as can be seen from table 3
, both analyses resulted in scale - h values exceeding 0.3 , implying that both scale solutions could be considered acceptable . however , closer inspection of the individual item - h ( hi ) values revealed that 5 items had hi values lower than 0.3 for the unidimensional model .
this was not the case in the second analysis : here , all hi values exceeded 0.3 .
tests for monotonicity available in msp5.0 indicated that this assumption was possibly violated for item 10 .
however , visual inspection of the summary irf for this item , using testgraf98 , did not show any violation .
the checks for double monotonicity did not show any violations . furthermore , the item ordering within the two subscales was comparable for the five age groups .
these curves depict the measurement precision for a person with a given score on the latent trait scale ( ) , which is estimated by the expected total score .
the graphs clearly show that the measurement precision for the two subscales is best at the lower levels of expected total score .
this observed difference of information at different levels of suggests that the two rumination subscales can not distinguish reliably between persons with moderately high and very high scores on the brooding and reflection subscales .
taken together , the factor structure reported in the english language version of treynor and colleagues has been replicated in the german translation and was independent of age .
significant age effects were found for both the brooding subscale ( f(4,293 ) = 5.63 , p = .000 , partial = .07 ) and the reflective pondering subscale ( f(4,293 ) = 5.93 , p = .000 , partial = .08 ) .
post hoc comparisons using tukey 's hsd test with an adjusted alpha level of .01 indicated that , on the reflective pondering subscale the oldest age group ( 63 years ) compared to the three age groups 24 years ( d = .77 ) , 2537 ( d = .76 ) , and 3850 ( d = .85 ) showed lower scores ( p < .001 ) and marginally significant lower scores compared to the second oldest age group of 5162 years ( d = .59 ) .
scores on the brooding subscale indicated statistically significant differences between the oldest age group ( 63 years ) and the youngest age group ( 24 years , d = .37 ) only . for both , brooding and reflective pondering subscales
overall , the oldest age group reported less ruminative thoughts , which was expressed in medium to large effect sizes in comparison to younger age groups . the final model for depression
brooding had a positive significant association with depression , which did not depend on age or sex .
the significant three - way interaction between age , reflective pondering , and sex indicates that the association between reflective pondering and depression was different for age , but only for one of the two sexes .
since men were used as the reference category , the significant two - way interaction between reflective pondering and age indicates that for men up to 24 years and 63 years and older , the association between reflection and depression was stronger than for men at the age of 25 to 37 ( positive value of standardized beta ) . for women ,
this is indicated by the significant three - way interaction of age , reflective pondering , and sex , which has a negative standardized beta value of approximately the same size as the positive value of the two - way interaction . the final model for life satisfaction
reflective pondering was not associated with life satisfaction and was , therefore , not included in the final model .
this effect was weaker for age groups 3850 years and 51 to 62 years compared to group 25 to 37 years , which is indicated by a significant negative standardized beta value for the two - way interactions between brooding and respective age groups .
significant main effects were also found for these age groups , indicating that participants aged 38 to 62 experienced a lower life satisfaction than participants aged 25 to 37 , regardless of sex or brooding .
to our knowledge , the present study is the first to investigate depressive ruminative response styles in different age groups in a cross - sectional design in general , and the association of rumination with depression on the one hand , and life satisfaction on the other . by comparing age groups this study builds upon previous work investigating the presence of perseverative thinking in exclusively young or old samples and previous research replicating the association between rumination and depression in older samples without differentiating between ruminative styles . up to now
life satisfaction as a cognitive judgment of well - being in relation to one 's own individual standards has not yet been investigated in relation to ruminative response styles .
prior to further analyses , however , a german translation of the 10-item version of the rsq was subjected to a nonparametric mokken scale analysis based on item response theory , which confirmed the factor assignment as previously reported by treynor and colleagues .
the factor assignment was identical in all age groups , the item - ordering in the msa comparable , demonstrating that the 10-item version of the rsq is adequate for younger and older samples alike .
results of the present study are in line with numerous earlier findings suggesting a stable association between ruminative response style and depression ( e.g. , [ 6 , 7 ] ) .
as expected , this association was significantly stronger for the subscale brooding as compared to reflective pondering and most pronounced for the youngest and the oldest age group .
the oldest age group showed remarkably low indicators of rumination ( i.e. , low brooding and low reflective pondering sores ) .
this association was pronounced in the youngest and oldest age group , supporting the concept of separate cognitive mechanisms for brooding and reflective pondering .
effects of age categories indicated a high burden of ruminative brooding in the youngest age group .
this finding was accompanied by high depression scores in young people , although their life satisfaction was not negatively affected in comparison to other age groups .
notably , reflective pondering in the oldest and youngest age group of men was more strongly associated with depression than for their female counterparts .
previous results suggest higher levels of ruminative behavior in women compared to men [ 4 , 5 , 7 ] , accounting for sex differences in the prevalence of major depression .
the present study suggests that the association between pondering and depression is not necessarily absent in male samples but depends on the life period in which it occurs .
the reasons for age- and sex - dependent associations between reflective pondering and depression have to remain unanswered at this point .
a promising approach could be to investigate age - dependent topics of reflection or brooding that might differ between the sexes .
we suggest that different periods over the life span and their particular cohorts are confronted with age - specific life events , challenges , and opportunities .
future research should also investigate potential determinants of the varying association between reflective pondering and depression at different life stages , for example , the actual content of reflection , physical and mental health status , and so forth .
it could be speculated , for example , that the shorter life expectancy of men together with the increased likelihood of health complaints in the oldest age group might change the content of reflective pondering towards more health - related thinking , thus increasing the likelihood of sex differences to emerge .
however , further speculations on the underlying processes and mechanisms accounting for these effects are beyond the scope and possibilities of this cross - sectional study .
these results have implications for cognitive therapies with older patients . whereas the special needs of young and adolescent persons have been discussed for many years and are now included in the curricula of clinical trainings , the specific requirements of psychological interventions for older people have only recently started to be taken into consideration .
research on good aging [ 5255 ] and its clinical consequences for cognitive behavioral psychotherapy as well as the ccmsc - model ( context , cohort , maturation , & specific challenges ; ) are examples of this ongoing development of paradigms .
the different ruminative behavior as reflected in the current self - reports may be due to the fact that cohorts are raised in specific historical contexts and therefore in their particular social and societal environments .
the further development of cohort - specific intervention requires a sound understanding of developmental aspects of cognitive function , particularly where risk factors for mood disorders are concerned .
the present study contributes to this understanding in a way that ruminative behavior appears to be less pronounced in older adults beyond retirement age and that this ruminative style is associated with depression and negative affect to a lesser extent , as it is at a younger age .
further research is needed to compare these findings with adult clinical samples or vulnerable groups with specific physical or psychological impairments and to identify the mechanisms underlying these stabilizing and protective processes .
these processes have been discussed in terms of goal adjustment and their age - related shifts [ 52 , 54 ] . at a stage of life when irreversible and uncontrollable events accumulate , accommodation of goals and a different weighing of personal priorities have been shown to provide the basis for the remarkable stability , resourcefulness , and resilience of aging adults .
one limitation of this study is the lack of health - related information that might have influenced the responses .
it was seen as not appropriate to ask personal questions regarding one 's health in the various settings .
thus , it can not be ruled out that fidelity of answering personal questions and social desirability brought forward in a questionnaire delivered by an unknown person is also influenced by the age or sex of the participant as well as the researcher and that these experimenter effects are age specific .
nevertheless , higher age is more likely to be associated with ill health , and thus health - related rumination would have worked against the hypothesis of lower rumination scores in high age .
the fact that this study is cross - sectional is seen as providing important information about possible , although not quantifiable , cohort effects that are expected to exist and to imply differential consequences for therapeutic approaches .
it can not be ruled out that the particular settings where participants were recruited for the current study implied certain self - selection biases .
nevertheless , a certain selection bias is unavoidable in studies investigating representative samples of the population at large , who do not have the possibility to draw a balanced sample ( e.g. , with the help of public authorities or opinion poll agencies ) but recruit participants in a random fashion .
subsequent studies should replicate the present results in other samples , or particularly chosen subsamples ( e.g. , age groups ) .
the presented results provide a replication of treynor 's and armey 's two - factor structure of a german translation of the rsq 's 10-item rumination subscale .
the dimensionality of brooding and reflecting could be confirmed in a mokken scale analysis based on nonparametric item response theory modeling , supporting earlier notions of distinct cognitive processes for reflective pondering and brooding .
previous findings on a positive relationship between depression and rumination were confirmed , as was the particular close association between brooding and depression .
this association was most pronounced for the youngest and oldest age group , although the absolute values indicated a relatively lower burden of rumination in the oldest age group .
life satisfaction was associated with brooding but not with reflective pondering . to our knowledge , this study is the first to investigate the association of ruminative styles and depression in different age groups .
the findings are in line with recent research on successful aging and data on vulnerability for psychological problems in adolescents .
the results suggest further that more research is needed on age - dependent effects of ruminative styles and their potential consequences for clinical psychological assessment and intervention . more generally , this study aims to inspire further research including comparisons of various life stages and requires more detailed investigations of the underlying mechanisms , particularly of age by sex interactions , which were beyond the possible scope of the present study .
given the rather low scores of rumination in the older age group , it could be speculated , for example , that interventions designed to reduce depression in older age might be more effective if they focused on behavioral activation rather than cognitive restructuring .
moreover , results on life - satisfaction indicated age - dependent associations between ruminative styles and satisfaction with life on the one hand and depression on the other . | rumination has been defined as a mode of responding to distress that involves passively focusing one 's attention on symptoms of distress without taking action .
this dysfunctional response style intensifies depressed mood , impairs interpersonal problem solving , and leads to more pessimistic future perspectives and less social support .
as most of these results were obtained from younger people , it remains unclear how age affects ruminative thinking .
three hundred members of the general public ranging in age from 15 to 87 years were asked about their ruminative styles using the response styles questionnaire ( rsq ) , depression and satisfaction with life .
a mokken scale analysis confirmed the two - factor structure of the rsq with brooding and reflective pondering as subcomponents of rumination .
older participants ( 63 years and older ) reported less ruminative thinking than other age groups .
life satisfaction was associated with brooding and highest for the earlier and latest life stages investigated in this study . |
the etude epidmiologique auprs des femmes de la mutuelle gnrale de leducation nationale ( e3n ) is a french prospective cohort study of 98,995 female members of a health insurance plan covering mostly teachers and teacher - spouses that began in 1990 and is the french component of the epic ( european prospective investigation into cancer and nutrition ) study .
participants return mailed questionnaires to update health - related information every 23 years , and a drug reimbursement claims database has been available since 2004 from their medical insurance provider ( mgen [ mutuelle gnrale de leducation nationale ] ) .
average follow - up per questionnaire cycle has been 83% , and loss to follow - up is < 3% . in 1992
, 86,164 participants responded to a questionnaire that included detailed information on secondhand tobacco smoke exposure .
we excluded current ( n = 12,611 ) and past smokers ( n = 27,061 ) and women with missing smoking status ( n = 5,072 ) ; with prevalent type 2 diabetes , cancer , or cardiovascular disease ( n = 3,161 ) ; and with no follow - up after 1992 ( n = 916 ) .
all participants signed an informed consent letter to comply with the french national commission for computerized data and individual freedom . in 1992
, participants were asked about parental smoking during their childhood ( yes , no , do not know ) . for frequency of exposure in childhood , participants were asked , during childhood , how often did you remain in a room with tobacco smoke ? ( never , do not know , rarely , occasionally [ some hours per week ] , a few hours per day , many hours per day ) . regarding their current exposure , participants were asked , currently , does your spouse ( or the person you live with ) smoke ?
finally , participants were asked to estimate their daily secondhand tobacco smoke exposure at home and outside the home separately by summing the time spent during the day in a room while someone was smoking ( 18 response categories from 0 to 16 h / day ) . as previously described ( 20 ) ,
3,496 type 2 diabetes cases were identified up to 2007 through information from several sources ( supplementary fig .
, potential cases were women who self - reported either type 2 diabetes , use of diabetic medications , or a hospitalization for type 2 diabetes in at least one of the eight follow - up questionnaires up to july 2005 ( n = 4,289 ) . among them , 2,315 were found to have at least one reimbursement claim for diabetic medications ( acarbose , carbutamide , glibenclamide , glibornuride , glicazide , glimepiride , glipizide , insulin , metformin , pioglitazone , repaglinide , or rosiglitazone ) and were classified as confirmed cases .
of the 1,974 remaining , 342 were validated through a supplementary questionnaire that assessed diagnosis date , symptoms , fasting or random glucose concentrations at diagnosis , current therapy , and the most recent values for fasting glucose and hba1c .
type 2 diabetes was confirmed if participants reported a glucose level at diagnosis above the world health organization recommendations ( fasting 1.26 g / l , random glucose 2.00
g / l ) , being on drug therapy for type 2 diabetes , or having a recent fasting glucose level of 1.26
date of diagnosis was defined as the date of first report of type 2 diabetes . as a second validation strategy
, we sent the supplementary questionnaire to 1,139 women who had filed at least once for reimbursement for diabetic medications between 1 january 2004 and 30 june 2007 and had not previously self - reported type 2 diabetes ; we confirmed type 2 diabetes in 458 . among the 405 women who did not respond to the supplementary questionnaire
, we confirmed type 2 diabetes in 338 who had filed for diabetic medication reimbursement two or more times . in the baseline questionnaire ( in 1990 ) , we obtained information on parental history of diabetes , education , body silhouette at age 8 , menopausal status , weight , height , physical activity , treated hypertension , and hypercholesterolemia .
weight and height were used to calculate the bmi ( defined as weight [ kg ] divided by height squared [ m ] ) .
regular moderate and vigorous physical activity were assessed with a validated physical activity questionnaire and transformed into weekly mets ( 21 ) .
dietary data were collected in 1993 with a previously validated self - administered diet history questionnaire ( 22 ) .
we classified participants as having no or at least one parent who smoked and assumed that participants who responded that they did not know the smoking status of their parents were not exposed .
participants were also classified into one of the following categories : never , rare , occasional , and regular childhood secondhand smoke exposure .
total daily adult secondhand smoke exposure was defined as the sum of secondhand smoke exposure at home and outside the home and was categorized as no exposure or < 1 , 11.9 , 23.9 , or 4 h
when one component was missing , exposure level was set to 0 . for home exposure ,
person - time at risk was calculated from the date of completion of the 1992 questionnaire to the date of diagnosis of type 2 diabetes , death , mailing of the last follow - up questionnaire ( 2005 ) , or date of last follow - up , whichever occurred first .
cox multivariate regression models with age as the time scale were fit to estimate hazard ratios ( hrs ) and 95% cis with the sas phreg procedure ( sas institute inc . ,
multivariate models were adjusted for parental history of diabetes , education ( less than high school , high school , and college or more ) , body silhouette at age 8 ( 1 , 2 , or 3 ) , childhood secondhand smoke exposure , treated hypercholesterolemia and hypertension , bmi ( continuously ) , menopausal hormone therapy ( premenopausal , ever use , never use ) , and physical activity level ( quartiles ) at baseline . in an additional multivariate model
, we included the following dietary factors : energy intake excluding alcohol ( quartiles ) , alcohol ( < 5 , 510 , 1015 , and 15 g / day ) and coffee ( 0 , 1 , 13 , and 3 cups / day ) intake , and processed red meat consumption ( < 1 , 13 , 35 , and 5 servings / week ) . variables to adjust for confounding were diabetes risk factors that could potentially cause or share a common cause with secondhand smoke exposure . to test for linear trend ,
we tested whether the relationship between secondhand smoke and type 2 diabetes risk differed by bmi ( < 25 or 25 kg / m ) by including a cross - product term of the median or ordinal value for each category of secondhand smoke exposure as a continuous variable and bmi as a dichotomous variable .
we compared models with and without the cross - product term by log - likelihood test .
the etude epidmiologique auprs des femmes de la mutuelle gnrale de leducation nationale ( e3n ) is a french prospective cohort study of 98,995 female members of a health insurance plan covering mostly teachers and teacher - spouses that began in 1990 and is the french component of the epic ( european prospective investigation into cancer and nutrition ) study .
participants return mailed questionnaires to update health - related information every 23 years , and a drug reimbursement claims database has been available since 2004 from their medical insurance provider ( mgen [ mutuelle gnrale de leducation nationale ] ) .
average follow - up per questionnaire cycle has been 83% , and loss to follow - up is < 3% . in 1992
, 86,164 participants responded to a questionnaire that included detailed information on secondhand tobacco smoke exposure .
we excluded current ( n = 12,611 ) and past smokers ( n = 27,061 ) and women with missing smoking status ( n = 5,072 ) ; with prevalent type 2 diabetes , cancer , or cardiovascular disease ( n = 3,161 ) ; and with no follow - up after 1992 ( n = 916 ) .
all participants signed an informed consent letter to comply with the french national commission for computerized data and individual freedom .
in 1992 , participants were asked about parental smoking during their childhood ( yes , no , do not know ) . for frequency of exposure in childhood , participants were asked , during childhood , how often did you remain in a room with tobacco smoke ? ( never , do not know , rarely , occasionally [ some hours per week ] , a few hours per day , many hours per day ) . regarding their current exposure , participants were asked , currently , does your spouse ( or the person you live with ) smoke ? ( no spouse ; no ; yes , occasionally ; yes , regularly ) .
finally , participants were asked to estimate their daily secondhand tobacco smoke exposure at home and outside the home separately by summing the time spent during the day in a room while someone was smoking ( 18 response categories from 0 to 16 h / day ) .
as previously described ( 20 ) , 3,496 type 2 diabetes cases were identified up to 2007 through information from several sources ( supplementary fig .
, potential cases were women who self - reported either type 2 diabetes , use of diabetic medications , or a hospitalization for type 2 diabetes in at least one of the eight follow - up questionnaires up to july 2005 ( n = 4,289 ) . among them , 2,315 were found to have at least one reimbursement claim for diabetic medications ( acarbose , carbutamide , glibenclamide , glibornuride , glicazide , glimepiride , glipizide , insulin , metformin , pioglitazone , repaglinide , or rosiglitazone ) and were classified as confirmed cases . of the 1,974 remaining ,
342 were validated through a supplementary questionnaire that assessed diagnosis date , symptoms , fasting or random glucose concentrations at diagnosis , current therapy , and the most recent values for fasting glucose and hba1c .
type 2 diabetes was confirmed if participants reported a glucose level at diagnosis above the world health organization recommendations ( fasting 1.26 g / l , random glucose 2.00
g / l ) , being on drug therapy for type 2 diabetes , or having a recent fasting glucose level of 1.26
date of diagnosis was defined as the date of first report of type 2 diabetes . as a second validation strategy
, we sent the supplementary questionnaire to 1,139 women who had filed at least once for reimbursement for diabetic medications between 1 january 2004 and 30 june 2007 and had not previously self - reported type 2 diabetes ; we confirmed type 2 diabetes in 458 . among the 405 women who did not respond to the supplementary questionnaire
, we confirmed type 2 diabetes in 338 who had filed for diabetic medication reimbursement two or more times .
in the baseline questionnaire ( in 1990 ) , we obtained information on parental history of diabetes , education , body silhouette at age 8 , menopausal status , weight , height , physical activity , treated hypertension , and hypercholesterolemia .
weight and height were used to calculate the bmi ( defined as weight [ kg ] divided by height squared [ m ] ) .
regular moderate and vigorous physical activity were assessed with a validated physical activity questionnaire and transformed into weekly mets ( 21 ) .
dietary data were collected in 1993 with a previously validated self - administered diet history questionnaire ( 22 ) .
we classified participants as having no or at least one parent who smoked and assumed that participants who responded that they did not know the smoking status of their parents were not exposed .
participants were also classified into one of the following categories : never , rare , occasional , and regular childhood secondhand smoke exposure .
total daily adult secondhand smoke exposure was defined as the sum of secondhand smoke exposure at home and outside the home and was categorized as no exposure or < 1 , 11.9 , 23.9 , or 4 h / day , with no exposure to secondhand smoke used as the reference category . when one component was missing , exposure level was set to 0 . for home exposure ,
person - time at risk was calculated from the date of completion of the 1992 questionnaire to the date of diagnosis of type 2 diabetes , death , mailing of the last follow - up questionnaire ( 2005 ) , or date of last follow - up , whichever occurred first .
cox multivariate regression models with age as the time scale were fit to estimate hazard ratios ( hrs ) and 95% cis with the sas phreg procedure ( sas institute inc . ,
multivariate models were adjusted for parental history of diabetes , education ( less than high school , high school , and college or more ) , body silhouette at age 8 ( 1 , 2 , or 3 ) , childhood secondhand smoke exposure , treated hypercholesterolemia and hypertension , bmi ( continuously ) , menopausal hormone therapy ( premenopausal , ever use , never use ) , and physical activity level ( quartiles ) at baseline . in an additional multivariate model
, we included the following dietary factors : energy intake excluding alcohol ( quartiles ) , alcohol ( < 5 , 510 , 1015 , and 15 g / day ) and coffee ( 0 , 1 , 13 , and 3 cups / day ) intake , and processed red meat consumption ( < 1 , 13 , 35 , and 5 servings / week ) . variables to adjust for confounding were diabetes risk factors that could potentially cause or share a common cause with secondhand smoke exposure . to test for linear trend ,
we tested whether the relationship between secondhand smoke and type 2 diabetes risk differed by bmi ( < 25 or 25 kg / m ) by including a cross - product term of the median or ordinal value for each category of secondhand smoke exposure as a continuous variable and bmi as a dichotomous variable .
we compared models with and without the cross - product term by log - likelihood test .
after a median follow - up of 13.4 years ( 466,133 person - years ) , we identified 795 incident cases of type 2 diabetes .
most of the exposure was paternal , with only 3% of participants reporting exposure to maternal smoking .
incidence was 155 per 100,000 person - years among participants who were not exposed to childhood secondhand smoke and 182 per 100,000 person - years among those with at least one parent who smoked , leading to an age - adjusted hr of 1.18 ( 95% ci 1.021.36 ) . adjusting for potential confounders that may also be intermediates ( education and body silhouette at age 8) yielded a slightly attenuated estimate ( multivariate hr 1.15 , 0.991.33 ) . in an additional analysis
, we excluded 284 women who did not know or did not report parental smoking status and were considered unexposed .
there was no association between intensity of childhood secondhand smoke exposure and type 2 diabetes risk .
relative to no or unknown exposure , multivariate hrs were 0.96 ( 0.811.14 ) for rare , 1.04 ( 0.851.27 ) for occasional , and 1.08 ( 0.871.33 ) for regular ( a few hours and many hours per day ) exposure ( p = 0.45 for trend ) .
the mean sd adult secondhand smoke exposure was 0.9 1.9 h / day ( 10th90th percentile 0.02.5 ) , home exposure was 1.6 2.4 h / day ( 0.04.0 ) , and exposure outside the home was 0.5 1.3 h / day ( 0.01.0 ) .
bmi increased with increasing categories of total adult exposure to secondhand smoke , and higher educational attainment seemed to be more common among women who reported exposure than among those who identified themselves as unexposed ; however , among the exposed participants , we observed an inverse relation ( table 1 ) .
age - standardized characteristics of the cohort for adult secondhand smoke exposure , type 2 diabetes incidence was 170 per 100,000 person - years for both participants who reported no exposure and those who reported exposure .
however , the age - adjusted hr comparing women with exposure to adult secondhand smoke compared with no exposure was 1.18 ( 95% ci 1.021.36 ) and the multivariate hr was 1.16 ( 1.001.34 ) .
when evaluating the dose of exposure , the age - adjusted hr was 1.89 ( 1.462.43 , p < 0.0001 for trend ) for women who reported 4 h / day of secondhand exposure compared with those who reported no exposure ( table 2 ) .
after adjusting for parental history of diabetes , education , body silhouette at age 8 , childhood secondhand smoke exposure , bmi , physical activity , menopause , hormone replacement therapy , treated hypercholesterolemia and hypertension , and alcohol , coffee , and processed red meat consumption , participants who reported 4 h / day of secondhand smoke had a statistically significant 36% higher rate of type 2 diabetes than those who reported no exposure to secondhand smoke ( 1.36 [ 1.051.77 ] )
. there appeared to be a direct dose - response relationship between daily secondhand tobacco smoke exposure and the rate of type 2 diabetes in the multivariate model ( p = 0.002 for trend ) ( fig .
, we excluded 276 women who started smoking during follow - up to account for the possibility that women who are exposed to secondhand smoke may be more likely to start smoking .
rate of type 2 diabetes according to daily hours of exposure to secondhand smoke multivariate adjusted hrs for type 2 diabetes according to adult secondhand smoke exposure ( p = 0.008 for trend ) .
secondhand smoke was calculated by summing exposure at home and outside the home and adjusted for parental history of diabetes , education , body silhouette at age 8 , childhood secondhand smoke exposure , hormone replacement therapy ( premenopausal , ever , never ) , treated hypertension and hypercholesterolemia , and bmi ( continuous ) and for physical activity measured in mets per week ( quartiles ) , total energy in kilocalories per day ( quartiles ) , alcohol intake in grams per day ( quartiles ) , processed red meat consumption in servings per day ( quartiles ) , and coffee consumption in cups per day ( quartiles ) .
the association between type 2 diabetes risk and adult secondhand smoke seemed to be restricted to exposure outside the home .
the multivariate hr comparing participants who reported 4 h / day of secondhand smoke exposure outside the home to no exposure was 1.45 ( 95% ci 1.012.07 , p = 0.001 for trend ) .
women who lived with a person who smoked seemed to have a higher rate of type 2 diabetes than those who did not ; however , results were not significant ( 1.10 [ 0.841.44 ] for living with an occasional smoker and 1.15 [ 0.951.39 ] for living with a regular smoker ) .
the hr comparing women in the highest exposure category of daily hours of secondhand exposure at home compared with no exposure at home was 0.94 ( 0.571.53 ) .
information on hours of secondhand smoke exposure at home was missing for 75% of the person - time .
estimates comparing extreme categories of exposure were slightly strengthened but nonsignificant when we conducted sensitivity analyses for total exposure restricted to participants for whom information was complete for both sources of exposure ( 1.52 [ 0.952.40 ] ) and for secondhand exposure at home where missing values were considered 0 ( 1.20 [ 0.811.77 ] ) .
the observed association between secondhand smoke and type 2 diabetes differed slightly according to bmi .
the hrs comparing extreme categories of exposure to total secondhand smoke exposure were 1.82 ( 95% ci 1.202.76 , p = 0.001 for trend ) for bmi < 25 kg / m and 1.36 ( 0.991.89 , p = 0.08 for trend ) for bmi 25 kg / m , but the test for heterogeneity was not statistically significant ( p = 0.28 )
. we also evaluated whether having had a parent who smoked resulted in differences in the association between secondhand smoke exposure in adulthood and type 2 diabetes .
we found no evidence of a statistical interaction ( p = 0.29 for the test for heterogeneity ) .
in a large prospective cohort of nonsmoking french women free of type 2 diabetes at baseline and followed for up to 15 years , type 2 diabetes rates increased with secondhand exposure in childhood and with increasing exposure to secondhand smoke in adulthood . for adult exposure , the association appeared to be restricted to exposure outside the home .
rats exposed to nicotine early in life have higher fat mass , insulinemia , and increased leptin levels in adulthood than do unexposed rats ( 10 ) .
thus , early nicotine exposure may program individuals for future adipocyte hypertrophy and insulin and leptin resistance .
additionally , exposure to environmental tobacco smoke results in chronic pancreatic inflammation in rats ( 9 ) and induces the production of interleukin-1 ( 23 ) in the lung .
this cytokine is believed to govern the pancreatic inflammation observed in type 2 diabetes that may result in -cell death and impair insulin production ( 24 ) .
a meta - analysis of 25 prospective cohort studies found a 44% higher rate of type 2 diabetes among smokers than among nonsmokers ( 8) .
however , evidence for the role of secondhand smoke in type 2 diabetes risk is limited . in the present study
, we observed a significant association between parental smoking and subsequent risk of type 2 diabetes .
we evaluated the sensitivity of the results to adjustment by a proxy of parental social status , the participants education , and body size at age 8 .
the association became borderline statistically significant ; however , these variables could potentially be intermediates . in cross - sectional analyses of children ( 12 ) and adolescents ( 25 ) , individuals exposed to environmental tobacco were four times more likely to be overweight than those who were not exposed . in a group of 10-year - olds participating in two birth cohorts , having a mother who smoked during pregnancy and being exposed to secondhand tobacco smoke in childhood
it is unlikely that our observation is the result of maternal smoking during pregnancy or lactation because only 3% of the participants with a parent who smoked reported that the parent was their mother .
thus , the present results may only reflect childhood secondhand smoke exposure and not exposure in utero . in a large prospective cohort of women with a 24-year follow - up in the united states , a borderline significant 16% higher rate of type 2 diabetes
was observed among nonsmokers who were regularly exposed to secondhand cigarette smoke than among women who reported not being exposed ( 19 ) . in a prospective study of young adults with a 15-year follow - up ,
passive never - smokers had a 35% higher rate of glucose intolerance relative to nonsmokers ( 17 ) . in a much smaller cohort of 4,442 korean never - smokers with a detailed assessment of frequency and duration of secondhand smoke at home and at work
, there appeared to be a linear relationship between daily hours of exposure and incidence of type 2 diabetes ( 16 ) .
compared with participants who declared not being exposed to secondhand tobacco smoke , the incidence of type 2 diabetes among those who declared exposure to 01 , 1.12 , 2.14 , and > 4 h / day was 34 , 32 , 44 , and 96% higher , respectively . our estimate for individuals exposed to 4 h / day was smaller than that observed in korea ( hr 1.36 ) .
residual confounding by omitted lifestyle variables in the korean study , like dietary factors , may partly explain the observed difference in estimates .
there are several explanations for the absence of an association between secondhand smoke exposure at home and type 2 diabetes in the present study . for a large proportion of participants ,
we were unable to assign secondhand exposure at home ; thus , the analysis may be underpowered to detect an association , and given the limited number of cases in certain categories , estimates are very unstable .
alternatively , the intensity of exposure may be much higher outside the home than within the home .
this explanation is consistent with a prior observation that secondhand smoke outside the home is associated with lung cancer , but no association was found with exposure at home ( 26 ) .
the major strengths of this study are its prospective nature , detailed report of secondhand smoke exposure , large sample size , and availability of type 2 diabetes risk factor information .
one limitation was the use of a nonvalidated self - reported assessment of secondhand smoke as the main exposure measure .
because in many environments secondhand smoke is ubiquitous , misclassification of secondhand exposure is common . in a group of people with asthma ,
the correlation between reported exposure and urine cotinine levels was 0.47 ( 27 ) . among nonsmokers in a cancer screening clinic , 76% reported exposure to secondhand smoke , whereas 91% had detectable levels of cotinine ( 28 ) .
in addition , secondhand smoke exposure was evaluated only at baseline , whereas the exposure level may have changed over time .
nevertheless , it is likely that the error introduced is random and independent of the outcome , which would result in an attenuation of the observed associations and may explain the null results with regard to intensity of childhood and at - home exposure to secondhand smoke .
additionally , we expected few , if any , false - positive results ; therefore , the bias introduced was probably minimal
. we can not rule out the possibility of confounding by unmeasured factors and residual confounding by risk factors for type 2 diabetes that were measured with error .
for example , parental social status may be a common cause of parental smoking and type 2 diabetes risk .
however , as mentioned previously , we were able to include in our analyses all the major lifestyle risk factors for type 2 diabetes , and we have previously shown the validity of these factors ( 21,22 ) .
selection bias may have been introduced by excluding from our analyses 5,072 women ( 6.2% of the cohort ) for whom information on smoking status at baseline was not available .
finally , the results may not be generalizable to men or non - caucasian populations .
however , the results are similar to those reported in an asian population comprising both sexes , indicating that the associations may not differ by race , ethnicity , or sex ( 16 ) . in conclusion
, this prospective analysis suggests that secondhand smoke exposure in childhood and adulthood is associated with a higher rate of type 2 diabetes .
smoke - free environments have proven to be relatively easy to implement and effective in the control of cardiovascular disease ( 2,3 ) .
curbing the worldwide type 2 diabetes epidemic requires extensive and lasting changes in public policy . limiting secondhand smoke exposure by providing smoke - free environments and improving compliance with smoking bans may be an important strategy . | objectivethe objective of this study was to evaluate the relationship between childhood and adult secondhand smoke and type 2 diabetes.research design and methodswe conducted a prospective cohort study among 37,343 french women from the e3n - epic ( etude epidmiologique auprs des femmes de la mutuelle gnrale de leducation nationale - european prospective investigation into cancer and nutrition ) who never smoked and who were free of type 2 diabetes , cancer , or cardiovascular disease at baseline in 1992 .
self - reported childhood secondhand smoke exposure was defined as having at least one parent who smoked .
adult secondhand smoke was defined as the sum of self - reported hours recorded at baseline of exposure to tobacco smoke from a spouse who smoked ( or domestic close contact ) and from outside the home.resultsbetween 1992 and 2007 , 795 cases of incident type 2 diabetes were identified and validated through a drug reimbursement dataset and a specific questionnaire .
women with at least one parent who smoked appeared to have an 18% higher rate of type 2 diabetes than women with parents who did not smoke ( age - adjusted hazard ratio 1.18 [ 95% ci 1.021.36 ] ) .
adult secondhand smoke exposure ( no exposure versus 4 h / day ) was associated with an increased rate of type 2 diabetes ( 1.36 [ 1.051.77 ] , p = 0.002 for trend ) after adjusting for parental history of diabetes , education , body silhouette at age 8 , childhood secondhand smoke exposure , physical activity , body mass index , hypertension , hypercholesterolemia , menopausal status and hormone use , alcohol intake , and processed red meat and coffee consumption.conclusionsthis prospective analysis suggests that secondhand smoke exposure in childhood and adulthood are associated with a higher rate of type 2 diabetes . |
a stroke results in a damaged state of the peripheral external nervous system arising from
the necrosis of nerve cells in specific brain areas , caused by the interruption of blood
supply to brain cells due to the blockage or rupture of blood vessels transporting oxygen
and glucose to the brain1 . though various
problems may be evident in the fields of exercise , sense , recognition , language and
perception , depending on the part , size and cause of this damage , its main symptom is
hemiplegia2 .
thus ,
balancing and gait training for hemiplegic stroke patients should reflect the motor skills
and cognitive function required in daily living dual tasks3 .
dual tasks fall into two main groups : motor dual tasks4 , which require performance of a motor task
and a postural control task at the same time ; and cognition dual task5 which require performance of a cognition task and postural
control task at the same time . both types of dual task
are noted as ways of training
patients with neurological damage to recover their motor control ability . up to the present ,
research has emphasized the role of recognition and concentration during
dual task performance for posture and gait control as a paradigm of motor learning6 . it is asserted that subjects need to
simultaneously perform motor tasks and high cognitive functions , as daily living frequently
requires the performance of several tasks simultaneously , and it has been reported that
stroke patients who could carry out local community ambulation , had difficulties in
simultaneously performing dual task while walking7 .
viewing the recent trend of studies of dual task training for stroke patients , we noticed
that they elicited an interaction between gait and cognitive task through dual task methods ,
by making patients carry out cognitive tasks such as hearing , viewing or language tasks ,
together with walking8 , and that most
research mainly focused on the analysis of gait variance the course of training with motor
dual tasks7 .
few studies had conducted
various dual task training methods in order to enhance the balance and gait abilities of
stroke patients , and little comparative research has been conducted of the extent of
improvement among the various dual task training methods .
accordingly , this research investigated the changes occurring in terms of balance and gait
abilities , when various dual task gait training were conducted for chronic ambulatory stroke
patients to investigate the efficacy of dual task training methods in order to suggest new
therapeutic interventions that would help chronic stroke patients to improve their daily
living function .
thirty - six chronic stroke outpatients who were receiving treatment during the period of
june to august at j general hospital in kyeong - gi province , were randomly selected as the
subjects of this research .
all the subjects were randomly assigned to 3 groups : the motor dual task gait training
( mdgt ) group of 12 subjects , the cognitive dual task gait training ( cdtg ) group of 12
subjects , and the motor and cognition dual task gait training ( mcdgt ) group of 12 subjects .
gait training for 8 weeks was conducted for all of these 3 groups . in the midst of training ,
one subject left the mdtg group due to hospital transfer one
subject made a decision not to
continue cdtg group training , and one subject was excluded from the analysis due to a poor
attendance rate of less than 80% in mcdgt group training .
before the intervention , subject s
general features , balance ability and gait ability were measured and recorded . to improve
the reliability of the results , one researcher gave an outline and demonstrated in person
the task performance and experimental order , one week prior to the preliminary measurements
for the smooth progress of training and for the prevention of accidents .
also , for the
purpose of enhancing inter tester reliability , the testers who were to carry out the same
test , were allowed to have sufficient discussion and prior education on the test procedures .
in order to prevent any selection bias ,
the subjects were randomly divided into 3 groups ,
and11 subjects in the mdgt group , 11 subjects in the cdgt group and 11 subjects in the mcdgt
group completed the training of 30 minutes exercise , 3 times a week , for 8 weeks .
subjects in the mdgt group were instructed to perform some motor tasks while continuously
walking on a treadmill .
while walking on the treadmill , they performed 5 types of motor
tasks : tossing up and catching a ball , rehanging loops on different hooks , doing up
buttons after unbuttoning7 , holding a
cup of water without spilling it and receiving and returning a cup of water. three
minutes were allowed for each task , so 15 minutes were allowed for the set of 5 tasks , and 2
sets of tasks were carried out by the subjects9 .
the subjects carried out the cognitive dual tasks while maintaining
their gait on the treadmill .
mathematical subtraction , verbal analogical reasoning , spelling words
backward10 , and counting
backward11.three minutes were allowed
for each task , so 15 minutes were allowed for the set of 5 tasks , and 2 sets of tasks were
carried out by the subjects .
they performed the set of motor dual tasks for 15 minutes and the set of cognitive
dual tasks for 15 minutes , while maintaining gait on the treadmill .
pasw statistics 18.0 program for windows was used in this research to carry out all the
statistical analyses . in order to compare gender , paretic
side and stroke type of the 3
groups , the test was used , and one - way anova was used to compare age , height ,
and weight , and to test the homogeneity of the dependent variables before training .
the
kolmorogov - smirnov test was used to test the normality of the data .
the paired t - test was
used to check the difference between pre- and post - treatment in each group . in order to
compare the difference depending on the type of therapy among the 3 groups , we used analysis
of covariance ( ancova ) , setting the group variable of 3 groups as the fixed factor , and the
post - training scores as dependent variables after having controlled for the pre - test score
of dependent variables as covariance .
the changes in balance and gait abilities between pre- and post- training are shown in
table 1table 1 .
comparison of balance and gait abilities within and among the three
groupsgroupvariablemdgtcdgtmcdgtmean sdmean sdmean sdnostipre28.70 7.8328.39 5.0728.76 3.69post19.01 6.9924.52 4.8212.95 2.19change9.68 2.17 3.87 1.70 15.81 4.17 ncstipre47.39 10.8447.21 9.6047.26 13.66post35.87 10.6043.35 8.6324.92 10.60change11.52 2.32 3.86 1.91 22.35 9.80 nowdipre8.32 1.368.30 0.988.82 0.74post7.01 1.127.14 0.696.22 0.40change1.31 0.56 1.15 0.77 2.60 0.61 ncwdipre10.13 2.1310.34 1.4610.47 0.86post7.42 1.449.22 1.337.31 0.66change2.71 2.16 1.12 0.77 3.15 0.74 frt(cm)pre22.56 7.8622.36 5.5522.27 6.13post26.14 8.0623.82 5.9127.82 6.32change3.57 0.50 1.45 3.56 5.55 1.13 tug(sec)pre19.28 9.5019.33 10.9919.45 7.92post15.35 6.6817.31 9.9015.75 7.73change3.94 3.532.02 1.873.69 2.31fsst(sec)pre21.17 9.3021.08 11.7021.07 7.73post18.92 8.8618.59 12.3416.16 7.34change2.25 1.67 2.49 2.12 4.91 3.16 10 m wt(m
/ s)pre0.85 0.380.93 0.480.89 0.38post0.98 0.490.96 0.511.26 0.54change0.13 0.110.03
0.040.38 0.216 min wt(m)pre223.00 82.33253.83 123.42246.68 97.00post242.07 82.33263.90 123.61277.66 89.94change19.07 0.8310.07 0.8330.98 30.45*expressed as p<0.05 .
no : normal eyes open ; nc : normal eyes closed ; sti : stability
test index ; wdi : weight distribution index ; frt : functional reach test ; tug : timed up
and go test ; fsst : four square step test ; 10 m wt : 10 m walk test ; 6 min wt : 6-min
walk test ; mdgt : motor dual task gait training group ; cdgt : cognition dual task gait
training group ; mcdgt : motor and cognition dual task gait training group .
significantly different compared with mdgt . significantly different
compared with cdgt . significantly different compared with mcdg .
the differences in the stability test index ( sti ) and the weight distribution
index ( wdi ) with the eyes open and closed conditions were between the significant within
each of the 3 groups , as were the differences in sti and wdi between pre - training and
post - training ( p<0.05 ) .
the
functional reach test distances of the 2 groups excluding the cdgtgroup , showed significant
differences between pre - training and post - training ( p<0.05 ) .
the differences in frt
between pre- and post - training showed a significant difference among the 3 group
( p<0.05 ) , and the extent of improvement in the mdgt group and mcdgt groups was
significantly greater than that of the cdgt group ( p<0.05 ) .
the differences in the timed
up and go test ( tug ) times between pre- and post training , were significant ( p<0.05 )
within each group , but there were not significant inter - group difference .
the differences in
the four square step test ( fsst ) between pre- and post- training were significant
( p<0.05 ) within each group and the extent of improvement in the mdgt group and mcdgt
group was significantly greater than that of the cdgt group ( p<0.05 ) .
the 10 m walk test
( wt ) was used to assess gait ability between pre- and post- training .
it showed significant
difference both within each group and among the 3 groups ( p<0.05 ) , and the mcdgt group
showed a significantly greater improvement than the other 2 groups ( p<0.05 ) .
the 6 min wt
showed significant differences both within each group and among the 3 groups ( p<0.05 ) ,
and the mcdgt and cdgt groups showed significantly greater improvement than the mdgt group
( p<0.05 ) . * expressed as p<0.05 .
no : normal eyes open ; nc : normal eyes closed ; sti : stability
test index ; wdi : weight distribution index ; frt : functional reach test ; tug : timed up
and go test ; fsst : four square step test ; 10 m wt : 10 m walk test ; 6 min wt : 6-min
walk test ; mdgt : motor dual task gait training group ; cdgt : cognition dual task gait
training group ; mcdgt : motor and cognition dual task gait training group .
significantly different compared with mdgt . significantly different
compared with cdgt . significantly different compared with mcdg
this research investigated how various dual task training methods influenced the balance
and walking abilities of chronic stroke patients who were capable of local community
ambulation .
the subjects were assigned to the mdgt , cdgt , and mcdgt groups , and the
difference between pre - training and post - training was compared within and among these 3
groups . in all the assessments of balance ability in this research , except the tug test improvement
in the mcdgt group was significantly greater than in the other 2 groups . in the tests of
walking ability ,
both the 10 m walk test and 6 min walk test , performed before and after the
training , showed significant improvements in the 3 groups , and the mcdgt group showed more
significant improvement than the other 2 groups .
there were differences in the sti ( stability test index ) and wdi ( weight distribution
index ) between pre- and post - training in each of the 3 groups and the mcdgt group showed
greater improvement than the other 2 groups ( mdgt group and cdgt group ) . in 3 different
types of balance training were performed by 21 elderly persons with balance impairments .
the
study of silsupadol et al.12 , the
postural sway of the group that carried out the single task together with the dual task ,
declined by 56% showing their balance ability was improved .
the result of our present
research agrees with the results of previous research , in that the performance of dual task
decreased postural sway and improved postural stability13 .
investigated how the degree of postural sway varied
with performance of a dual task while walking , using patients who had had hemiplegia for 16
months on average as subjects .
they reported that the degree of postural sway decreased
while the patients were carrying out a dual task .
this research result of hyndman et
al.14 is also consistent with our
present findings . in the frt ( functional reach test ) , the mdgt group and mcdgt group showed improvements
after training , and they showed greater improvement than the cdgt group .
riley et al.15 reported that postural sway was corrected
better when patients performed a dual task of just keeping in contact with a curtain without
pulling it , than when they carried out a single task of balance .
their result is supported
by the research of wulf and prinz16 , who
concluded that the external focus of attention , concentrating on the result of the movement ,
is more effective than the internal focus of attention , concentrating on the movement
itself .
silsupadol et al.12 conducted a study in
which healthy elderly subjects performed dual tasks 3 times a week for 4 weeks .
the subject
performed single tasks such as standing up , standing up with their eyes closed , tandem
standing , standing on a moving surface , standing while holding a cup , catching balls , and
walking forward and backward , together with other space perception tasks .
silsupadol et
al.12 suggested that dual task training
improved the tug times , which is consistent with the result of our present research .
cheng
et al.17 reported that they found the
difference of vertical ground reaction force between the two lower limbs was lessened in the
action of standing up , after patients had performed a training course in which they repeated
the move of standing up with symmetrical posture .
sahrmann18 reported the position of the trunk relative to the pelvis of stroke
patients is changed because the muscles are asymmetrically shortened by neurological
injuries , and the shortened muscles are more easily mobilized than the stretched
antagonistic muscle on the unaffected side , so the level of tension of the shortened muscle
increased .
all the groups showed significant improvements in the four square step test , which is
designed to assess the balance ability of chronic stroke patients .
the mdgt group and the
mcdgt group showed significantly greater improvements than the cdgt group .
we consider this
result is consistent with that of reqnaux et al.19 , who reported attention was allocated more to the execution of
exercise than to cognitive performance when stroke patients performed tasks of differing
levels of difficulty while walking on a treadmill .
when the gait speed was compared among the 3 groups in the 10 m wt , which is designed to
measure the gait competence of chronic stroke patients , the mcdgt group showed significantly
greater improvement than the other 2 groups .
this result can be construed as agreeing with
the results of yang et al.7 , who reported
that subjects exhibited a significant improvement in gait speed after performing the 3 motor
tasks of simple walking , walking with buttoning task , and walking with the task of carrying
a cup on a tray .
their subjects were a group of 15 normal adults , a group of 15 stroke
patients with some difficulty with local community ambulation , and a group of 15 chronic
stroke patients with no difficulty with local community ambulation . in the 6-min
walk test , all of the groups exhibited significant improvements after
performing the training . in the research of salbach
et al.20 , 44 stroke patients were assessed in terms of gait ability after
they had practiced functional tasks for 6 weeks and their walking distance increased from
209 to 249 m after the intervention .
dean et al.21 asked an experimental group of stroke patients to perform
strengthening exercises for the paretic lower limb together with various functional tasks ,
and a control group of stroke patients to perform strengthening exercises mainly for the
upper limbs , and assessed the outcome with a circuit walking task .
they21 investigated , and found that the experimental group s gait
ability improved more than the control group s .
our present research showed that the extent of improvement was greater in the mdgt group
than in the cdgt group after the training .
the correlative between cognition and motor
functions has been the focus of research on dual task , as it is essential for understanding
how the recovery of motor control occurs after injury to the central nervous system8 .
morioka et al.22 asked healty individuals to perform a single task of
maintaining a standing posture on a force plate , and to perform a motor dual task of
maintaining posture while performing a cognitive dual task of a mathematic calculation .
they
reported that the subject s postural sway incresed during performance of the cognitive dual
task , a mathematical calculation , and there was no direct relation with the maintenance of
correct posture , while postural sway decresed during the performance of the motor dual task
for which the subjects were required to hold a tray with cups of water on it .
they further
noted that the subjects consciously tried to hold the tray horizontally while they performed
the motor task of holding the tray with cups of water on it , and that this conscious effort
reduced the postural sway .
their findings were confirmed by vereijken et al.23 , who reported that the internal focus of
attention , concentrating on the movement itself , during the performance of a motor task
inhibits self - operating postural control due to conscious control of the posture , while the
external focus of attention , concentrating on the result of the movement during the motor
task , promotes self - operating postural control .
our present research could not thoroughly exclude the influence of subjects daily living
activities on their balance and gait because it was not possible to entirely control the
experimental subjects daily living activities , and this is a limiting factor of this
research .
additionally , the generalization of our results should be performed with care as
the number of subjects in this research was limited and the subjects of this research
comprised only ambulatory chronic stroke patients .
we suggest that future research should investigate the
effects of dual motor task gait training for patients with neurological deficit from various
viewpoints , addressing the limitations of the present study .
this research was conducted to examine whether various kinds of dual task gait training
could improve the gait and balance abilities of chronic stroke patients .
the subjects ,
ambulatory chronic stroke patients , were , divided into 3 groups : the mdgt group , the cdgt
group , and the mcdgt group .
they performed the prescribed training for 30 minutes , 3 times a
week for 8 weeks .
after the training , a comparison of the improvement of gait and balance
abilities was made within and among the 3 groups .
all of the 3 groups showed improvements ,
especially the mcdgt group which exhibited the greatest improvements in gait and balance
abilities among 3 groups .
this study demonstrated that mcdgt was more effective at improving gait and balance
abilities than mdgt or cdgt , and it can be recommended as the most efficient training
forchronic stroke patients capable of local community ambulation . | [ purpose ] this study examined the effects of various dual task gait training methods
( motor dual task gait training , cognitive dual task gait training , and motor and cognitive
dual task gait training ) on the balance and gait abilities of chronic stroke patients .
[ subjects and methods ] thirty - three outpatients performed dual task gait training for 30
minutes per day , three times a week , for eight weeks from june to august , 2012 .
balance
ability was measured pre - and posttest using the stability test index , the weight
distribution index , the functional reach test , the timed up and go test , and the four
square step test
. gait ability was measured by the 10 m walk test and a 6 min walk test
before and after the training .
the paired t - test was used to compare measurements before
and after training within each group , and anova was used to compare measurements before
and after training among the groups .
[ results ] comparisons within each group indicated
significant differences in all variables between before and after the training in all
three groups .
comparison between the groups showed that the greatest improvements were
seen in all tests , except for the timed up and go test , following motor and cognitive dual
task gait training .
[ conclusion ] in a real walking environment , the motor and cognitive
dual task gait training was more effective at improving the balance and gait abilities of
chronic stroke patients than either the motor dual task gait training or the cognitive
dual task gait training alone . |
the clinical aspects of asthma are paroxysmal respiratory distress , recurrent cough , wheezing , and chest tightness .
park reported that acute asthma reversibly increases lung compliance and total lung capacity ( tlc ) .
the authors confirm anecdotal evidence of a smaller horizontal curve of the ribs as a characteristic finding on chest radiographs of asthma patients .
since the first rib is relatively short and the 12 rib is easily bothered by shadow of the diaphragm and abdominal soft - tissue ; the author chose the sixth rib for calculation of arc . because the number of ribs is 12 in each one side of chest , we choose the six ribs and using a chest radiograph of an asthma patient [ figure 1 ]
, we drew a line ( ab ) horizontally through the middle point of the six ribs , and point c was defined as the crossing point where the six ribs reaches the thoracic cage .
this was repeated for a non - asthma patient [ figure 2 ] , and we found the arc to be larger than that of the asthma patient .
we then collected the same number of cases of asthma and non - asthma patients from a medical center in northern taiwan , and measured the arc for statistical analysis .
ab was drawn horizontally through the middle point of the sixth rib ; point c is where the sixth rib reaches the thoracic cage .
the angle between the lines ab and ac was defined as the angle of rib curve a chest radiograph of a non - asthma patient .
we performed a retrospective review of 57 cases of asthma who were admitted between january 2011 and february 2011 , and 57 non - asthma patients who visited our emergency department without chest complaints for analyses .
asthma in the study subjects was defined as discharged with a diagnosis of asthma from chest ward and labeled by a pulmonologist .
non - asthmatic patients were randomized selected from emergency department in northern taiwan medical center without chest complaints and shortness of breath .
exclusion criteria in non - asthmatic patients included presentation as dyspnea or any other chest complaint in the emergency department .
the diagnosis of the 57 non - asthma patients admitted to the emergency department in northern taiwan medical center from march 1 , 2011 to march 10 , 2011 included gastroenteritis ( n=11 ) , trauma ( n=9 ) , urinary tract infection ( n=4 ) , depression ( n=4 ) , drug overdose ( n=3 ) , rhinitis / pharyngitis / tonsillitis ( n=3 ) , intracranial hemorrhage ( n=3 ) , headache / dizziness ( n=3 ) , abdominal pain ( n=2 ) , maylagia / neuralgia ( n=2 ) , upper gastrointestinal bleeding ( n=2 ) and infectious diarrhea , pneumonia , constipation , pelvic inflammatory disease , stroke , appendicitis , hyponatremia , convulsions , pancreatitis , hypertension and urolithiasis in one case each .
we measured the arc of each case and analyzed the data using the spss statistical software ( spss for windows , version 11.0 , spss inc . , and chicago , il ) .
the document number for pulling and reading medical records of the asthma patients is 2511243 .
the student 's t - test was used for statistical analyses , and significance was set at a p value of less than 0.05 .
the asthma group consisted of 15 males and 42 females with a mean age of 48.1 years ( sd 11.8 , range : 31 - 64 years ) .
the non - asthma group consisted of 30 males and 27 females with a mean age of 35.2 years ( sd 12.2 , range : 20 - 61 years ) .
the arc was smaller in asthma patients than in non - asthma patients ( 10.7 4.7 vs. 14.3 4.7 , p < 0.0001 ) . in the asthma group , the mean male arc was smaller than the mean female arc ( 8.4 4.1 vs. 11.5 4.7 , p = 0.026 ) ; however , there was no statistical difference in gender in the non - asthma group ( p = 0.405 ) [ table 1 ] [ figure 3 ] . in asthma and
non - asthmatics patients , age is not significantly related to arc ( p = 0.714 , and p = 0.107 ) [ figure 4 ] . basic data and comparisons of age and angle of rib curves of the asthma and non - asthma groups ( ) = n , = significant statistical difference comparisons of angle of rib curves ( arcs ) in different groups by asthma , non - asthma , and gender . asthma male have the smallest arc than other group ( ) = n in asthma ( blue spots ) and non - asthmatics patients ( red spots ) , age is not significantly related to angle of rib curve ( p=0.714 , and p=0.107 )
asthma is commonly seen in daily clinical practice . it is a systemic disease that involves inflammation of the pulmonary airways and bronchial hyperresponsiveness leading to the usually reversible clinical symptom of a lower airway obstruction . in acute episodes of asthma ,
hyperacute asthma was so - called acute respiratory failure within 3 h after acute exacerbation of asthma .
physiologically , bronchial hyperresponsiveness is documented by decreased bronchial airflow after bronchoprovocation with allergens such as cold air , respiratory tract infection , and cigarette smoke .
the gross pathology of asthmatic airways displays lung hyperinflation , smooth muscle hypertrophy , mucosal edema , and mucus gland hypersecretion .
acute reversible increases in tlc do occur during exacerbations of asthma was described in the year of 1990 .
there was a significantly smaller arc in the asthma group than the non - asthma group , and a smaller arc in the males in the asthma group compared to females .
the lesser curving of the ribs in asthma may results from the hyperinflation in asthma patients . in asthmatic patients , inspiratory with continuous outward motion of the upper anterior rib cage produced a horizontal aligned ribs in chest radiography . in non - asthmatics
expiration , the respiratory muscles are completely relaxed , but this is not the case in patients with asthma .
therefore , a smaller arc may be the anatomic consequence of asthma rather than the cause .
female asthmatic patients have significantly lower strength of inspiratory and expiratory muscle than male , this related to this study 's finding of smaller arc in male asthmatic patients than female 's .
lesser the hyperinflation associated with bronchoconstriction , lesser is the end - expiratory volume in the rib cage leading to larger arc .
we all know that diagnoses of asthma including evaluation for reversibility of airflow obstruction and bronchial hyperresponsiveness , not depending on radiographic trait of chest radiographs
. however , this study applied an interesting method and glance in raising suspicion of asthma patients .
this is the first study to report that the ribs of asthma patients are less sloped than in patients without asthma . in the opinion of the author ,
this trait of the chest radiograph can help a physician arouse a suspicion of bronchial asthma in day to day clinical practice . | objectives : a novel and interesting observation is that ribs go less sloped in asthma patients radiograph than non - asthmatics people . the aim of this study was to investigate whether a smaller horizontal curve of the ribs is a special trait on chest radiograph of asthma patients.materials and methods : a retrospective review of the chest radiographs of 57 cases of asthma who were admitted between january 2011 and february 2011 , and 57 non - asthma patients was performed .
chest radiographs were examined and lines drawn horizontally through the middle point of the sixth rib , and to where the sixth rib reaches the thoracic cage .
the angle between these two lines was defined as the angle of rib curve ( arc ) .
the arcs were then compared between groups using the student 's t-test.results:the mean arc was smaller in asthma patients than in non - asthma patients ( 10.7 4.7 vs. 14.3 4.7 , p < 0.0001 ) . in the asthma group ,
the mean male arc was smaller than the mean female arc ( 8.4 4.1 vs. 11.5 4.7 , p = 0.026 ) ; however , there was no statistical difference in gender in the non - asthma group ( p = 0.405).conclusions : this is the first study to report that the ribs of asthma patients are less sloped than in patients without asthma .
this photographic trait may be helpful in daily practice for suspecting a diagnosis of bronchial asthma . |
tobacco use is a leading public health problem all over the world with 82% of the world 's 1.1 billion smokers residing in low and middle income countries and where , in contrast to the declining consumption in high - income countries , tobacco consumption is on the rise .
tobacco consumption has overall been a major contributor to deaths due to circulatory diseases , pulmonary and malignant diseases in india .
smoking also increases the incidence of clinical tuberculosis , is a cause of half the male tuberculosis deaths in india , and of a quarter of all male deaths in middle age .
information on prevalence of tobacco use in india is available from surveys carried out in general community . according to the national cross - sectional household survey
, india has more than 200 million tobacco consumers ; however , prevalence of smoking and tobacco chewing varies widely between different states , and has a strong association with individual 's socio - cultural characteristics .
a recent nationwide study on smoking and mortality in india estimated that smoking in persons between the ages of 30 and 69 years is responsible for about 1 in 20 deaths of women and 1 in 5 deaths of men , totaling to 1 million deaths per year .
study of smoking pattern among middle age and elderly has received poor attention despite its proven implications on health .
the present study was thus carried out to determine :
prevalence and correlates of tobacco smoking among persons aged 30 years and above in a resettlement colony of delhi.level of awareness regarding hazards of tobacco smoking.quitting behavior among smokers and factors influencing such behavior .
prevalence and correlates of tobacco smoking among persons aged 30 years and above in a resettlement colony of delhi .
from previous study , the prevalence of current smoking in delhi was 23.2% , which would require a sample size of at least 595 subjects to estimate the prevalence of current smoking with 15% relative precision and with 95% confidence .
this cross - sectional study was conducted in gokulpuri , a resettlement colony in new delhi , india during may 2007 - april 2008 .
the study included persons aged 30 years and above residing in the study area at the time of survey .
the area has 4 blocks a , b , c , d with a total population of 22100 ( 5402 , 5222 , 5792 , 5684 respectively in the 4 blocks ) .
there are 4041 households and 2404 houses ( 568 , 576 , 643 , and 617 houses , respectively , in blocks a , b , c , d ) .
every 4 house was selected by the systematic random sampling method in each of the 4 blocks of the area . from each of the selected houses , one household having person(s ) 30 years and above age
if the house had more than one household with persons above 30 , lottery was drawn to select the household .
if there was no person above 30 in house visited , the immediate next house was chosen . in the selected household ,
all the persons above 30 years who were willing to participate and giving written informed consent were included in the study .
a total of 600 households ( 142 , 144 , 160 , and 154 , respectively , in the 4 blocks ) were selected , and 955 individuals were approached . out of these , 911 persons were interviewed ( 211 , 227 , 238 , and 235 persons in 4 blocks , respectively ) .
the socio - demographic variables collected were age , sex , educational attainment , and occupation of the participant and income of the family .
they were classified as current smokers : smoked regularly for within 1 month prior to examination ; non smokers : never smoked or occasionally smoking ; ex - smokers : stopped more than 1 month prior to examination ; and ever smokers comprising of current and ex - smokers .
details on the different forms of smoking used , including cigarette and bidi and hukkah as well as on the numbers smoked per day , were obtained from the smokers .
pack years were calculated from the average number of cigarettes and/or bidis smoked per day ; 1 pack year taken as smoking 20 cigarettes or 80 bidis for 1 year .
participants were asked to stand still on scale with face forward , and place arms on the sides of the body .
before the reading was taken , the participant was requested to have feet together , heels against the wall , knees straight , and look straight ahead and a point corresponding to height was marked on the wall using a hard cardboard . the distance between floor and the point
data was coded , analyzed , and statistically evaluated using spss ( statistical package for social sciences , version 16.0 ) .
odds ratios ( or ) , 95% confidence interval ( 95% ci ) , and p - values were calculated for each predictor variable .
multivariate logistic regression was performed to determine the independent predictors of current smoking by including variables significant at a level p 0.2 in univariate analysis .
further information was given to persons who consented to take part in the study , and the participants were free to leave the study any time if they desired to do so .
out of the 955 people approached , 911 consented to participate in the study giving a response rate of 95.3% .
a total of 911 individuals participated in the study with equal representation of both males and females .
the age of the participants varied from 30 to 90 years ( range - 60 years ) .
participants aged 30 - 59 years constituted 83.8% of the study population . there was no difference in the age - wise distribution of males and females up to age of 69 years ( = 2.40 , p = 0.49 ) .
more than half the population was either illiterate or only primary school pass [ table 1 ] .
socio - demographic characteristics of the study population in all , 224 participants were found to be currently smoking , giving an overall prevalence of current smoking to be 24.6% ( 95% ci 21.90 - 27.49 ) in the study population .
history of ever smoking was reported by 287 ( 31.5% ) participants ( 95% ci 28.57 - 34.59 ) [ table 2 ] .
distribution of study participants according to smoking status among current smokers , majority 198 ( 88.4% ) smoked bidi exclusively , 18 ( 8% ) smoked cigarette exclusively , and 8 ( 3.6% ) smoked a combination of bidi and hookah / cigarette .
with respect to the amount of tobacco smoke exposure in ever smokers , most 116 ( 40.4% ) had less than 2.5 pack year exposure , 78 ( 27.2% ) had an exposure between 2.51 - 6.25 pack years , 66 ( 23% ) between 6.26 - 13.50 pack years , whereas 27 ( 9.4% ) had more than 13.5 pack year exposure . on an average
more male participants were found to be currently smoking 184 ( 40.1% ) in comparison to females ( 8.8% ) , and the association between tobacco smoking and gender was statistically significant ( p < 0.001 ) [ table 2 ] .
smoking showed an increasing trend with an advancing age in both males and females [ table 3 ] .
on univariate analysis , smoking was found to be significantly associated with advancing age , primary and middle education , lower socio - economic status , unskilled semi - skilled and skilled occupation , and lower bmi . on multivariate analysis , the factors , which were found to be significantly associated with current smoking , were : male sex ( or = 5.74 ( 95% ci 2.86 - 11.49 ) ) , advancing age ( or = 1.99 ( 95% ci 1.25 - 3.17 ) for age 40 49 years , ( or = 2.57 ( 95% ci 1.66 - 3.99 ) for 50 years and above ) , lower education ( or = 2.07 ( 95% ci 1.27 - 3.38 ) ) for up to middle school , ( or = 2.36 ( 95% ci 1.30 - 4.27 ) for illiterate ) , skilled occupation ( or = 2.54 ( 95% ci 1.23 - 5.24 ) ) , lower socio - economic status ( or = 2.83 ( 95% ci 1.49 - 5.36 ) ) , and low bmi ( or = 2.10 ( 95% ci 1.33 - 3.32 ) ) [ table 4 ] .
there was an increasing trend of smoking with increasing age ( 2tr = 22.9 , p < 0.001 ) , decreasing bmi ( 2tr = 23.68 , p < 0.001 ) , and low socio - economic status ( 2tr = 9.82 , p = 0.0017 ) .
prevalence of current smoking among males and females according to socio - demographic factors and bmi multiple logistic regression analysis to evaluate factors associated with smoking of all participants in the study , 585 ( 64.2% ) were aware of the hazards of tobacco smoking .
current smokers 69.6% ( 156 out of 224 ) had better knowledge than non - smokers ( 62.4% ) .
most of the respondents believed that smoking increased the chances of tuberculosis , respiratory problems , and cancer [ table 5 ] .
participants awareness regarding hazards of tobacco smoking of the 287 ever smokers , 63 ( 21.9% ) had quit smoking in the past due to some reason .
17 ( 5.9% ) participants stopped smoking for less than a year , whereas majority 46 ( 16% ) had quit for more than a year . among the 63 successful quitters , 17 ( 27% )
did not have a specific reason to quit and did so by self offered vow to quit .
majority 35 ( 55.5% ) , however , quit smoking due to the initiation of health problems , which included respiratory problems ( 36.5% ) like diagnosed cases of chronic obstructive pulmonary disease , exacerbation of asthma , chronic cough , and breathlessness ; heart disease ( 3.17% ) and other illnesses ( 15.8% ) . apart from these , other factors contributing to quitting were social or familial pressure , either by spouse / other family member(s ) in 8 ( 12.6% ) cases and awareness regarding hazards of smoking in 3 ( 4.7% ) cases .
literate smokers were more likely to quit than illiterate smokers ( 60.3% vs 39.7% ) .
the purposes of this population - based study were to estimate the prevalence and correlates of current smoking among middle - aged and elderly people and to assess their knowledge and quitting behavior .
only few surveys have been conducted in delhi with the objective of estimating the prevalence of tobacco smoking , especially among these age groups .
national survey on smoking has shown wide variations between urban and rural areas , age , gender , education , and other socio - demographic variables across the country . in the present study ,
the prevalence of current smoking among people aged 30 years and above in an urban resettlement colony of delhi was estimated to be 24.6% , and this was much higher among males ( 40.1% ) than females ( 8.8% ) .
similar findings had been reported from delhi by chhabra et al . in a clustered community - based study in 2001 with about 50% of adult males between 30 - 60 years found to be current smokers , and very few female subjects admitting to smoking .
the overall prevalence of smoking among 4141 persons , aged 18 years and above , belonging to different socio - economic strata , was estimated to be 23.2% ( males 39.1% and females 2.6% ) .
other studies in delhi among persons aged 15 years and above have also shown a higher level of smoking habit among males ( 23.9% to 29.5% ) in comparison to females ( 1.8% to 4.2% ) .
recently , a survey in urban area of chennai also reported a 38% prevalence of smoking among males aged 35 - 69 years in the city .
one of the important factors for low prevalence of smoking among females was social unacceptability .
there was an increasing trend of smoking with an increasing age being higher among older age groups compared with the younger ones .
the prevalence of smoking in both the sexes increased with age leveling off after 70 years of age .
similar gender differences in smoking and increasing trends with age have been reported earlier in delhi , on a national level , and in zambia .
majority of the smokers in the present study preferred bidi ( 89% ) and few smoked cigarettes ( 8% ) .
bidi is preferred by the study population due to economic reasons as it is much cheaper than cigarette . on an average ,
similar findings have been reported by chhabra in delhi with 60% smokers above 18 years age smoking bidi .
a multi - centric study in delhi , kanpur , chandigarh , and bangalore also found that most ( 51.7% ) of the urban smokers smoked bidis with an average consumption being 12.4 bidi / cigarette per day . in the present study ,
tobacco use was highest amongst the illiterates wherein 54% and 12% of illiterate males and females respectively smoked tobacco .
other studies by rani and chhabra have also shown lower literacy level to be a strong predictor of smoking .
we also found 3-fold increased risk of smoking among people with low socio - economic status .
studies in the past have already suggested that the poor are 8 - 10 times more likely to smoke bidis .
similar trends have been observed in delhi by chhabra and on a country wide basis by jindal , and the national cross - sectional survey .
interestingly , we found that overweight or obese people were less likely to be current smokers than lean people .
overall , the study highlights that smoking is a significant problem among males who are lesser educated and belong to low socio - economic class . with regard to awareness regarding hazards of tobacco smoking , two third of the people in this study
however , there were huge gaps in knowledge , and majority participants knew very little of the wide implications of tobacco use on health . added to it , a sizable population did not have any knowledge of impact of tobacco on health .
poor literacy status has been found to be associated with poor awareness of the health hazards of tobacco consumption , increased likelihood of exposure to conditions favoring initiation of smoking and chewing of tobacco , and higher overall risk taking behavior . in the present study , around 21.9% of ever smokers
had quit smoking . among those who quit , majority did so due to their health problems . among 11000 ever smokers of 15 years and above age , jindal had reported a quit rate of 10% with health problems being an important reason for abstinence .
it is a matter of concern to find high levels of smoking , especially among the illiterate and poor people .
this is bound to increase the disease burden from chronic illnesses apart from communicable and nutrition - related diseases in these vulnerable groups .
smoking has also shown a rising trend with age emphasizing that initiation into the habit may occur at any age and not just among young people .
this implies that tobacco control policies will have to focus on almost all age groups up to the 50 plus age . in this
regard , health education can play a pivotal role and can have a lasting impact on reduction of tobacco smoking by improving awareness levels of the population .
this can be done through mass media and through school and community - based education programs . in view of the limited knowledge among people regarding health implications of tobacco use
, there is need to design appropriate health education material for illiterates and poorer sections of the population and also to elaborate the scope of warning labels on tobacco packs with focus on other potential hazards of smoking like heart disease , hypertension , chronic lung disease , and infertility as well rather than just focusing on cancer .
the association between smoking and body mass index should be explored further , so that an appropriate intervention can be designed that addresses both smoking and nutrition .
the survey was done in urban resettlement colony of delhi , and hence the results can only be generalized to the sampled population .
our survey was cross - sectional , and smoking status was by self - reporting .
therefore , some participants may have under - reported their smoking habit . moreover , due to the existing social taboo about tobacco use , some female participants , in particular , might not have reported their smoking habit . in spite of these limitations
the survey was done in urban resettlement colony of delhi , and hence the results can only be generalized to the sampled population .
our survey was cross - sectional , and smoking status was by self - reporting .
therefore , some participants may have under - reported their smoking habit . moreover , due to the existing social taboo about tobacco use , some female participants , in particular , might not have reported their smoking habit . in spite of these limitations | aim : to assess the prevalence and correlates of current smoking , awareness of hazards , and quitting behavior among smokers 30 years and above.materials and methods : study design : cross - sectional ; setting : gokulpuri , a resettlement colony in east delhi , india ; sample size : 911 , persons aged 30 years and above using systematic random sampling ; study tools : semi - structured questionnaire.results:prevalence of current smoking was found to be 24.6% ( 95% ci 21.90 - 27.49 ) .
majority 198 ( 88.4% ) of current smokers smoked bidi exclusively , and on an average 13.5 bidi / cigarette were smoked per day .
multivariate analysis showed the factors associated with current smoking as male sex , advancing age , illiteracy , skilled occupation , low socio - economic status , and low bmi ( p < 0.001 ) .
64.2% were aware of the hazards of smoking .
63 ( 21.9% ) had quit smoking in the past , majority due to the health problems .
low educational status was associated with poor hazard awareness and quitting behavior.conclusion:smoking is a significant problem among poor and illiterate males , shows an increasing trend with an advancing age and is directly associated with skilled occupation and low bmi .
there are significant gaps in knowledge regarding hazards of smoking . |
s. paratyphi b dt+ with the resistance phenotype apcmsmspsutc had been isolated sporadically in various states of australia since 1997 , and initial surveys showed a potential association with ownership of home aquariums ( d. lightfoot , unpub . data ) . in 2000 , multidrug - resistant s. paratyphi b dt+ with an identical phage type ( reaction does not conform [ rdnc ] ) , designated here as aus2 , and the same drug - resistance profile ( apcmsmspsutc ) was isolated from humans with gastroenteritis and from fish tanks in the homes of 2 infected patients ( table ) . in 2003 and 2004 , 13 cases of apcmsmspsutc s. paratyphi b dt+ were investigated by state and commonwealth health departments , and all were associated with home aquariums containing tropical fish ( j. musto et al .
11 cases were phage type rdnc aus3 , 1 was phage type 1 var 15 , and 1 was phage type 3b var .
water and gravel were collected from the domestic aquariums of 5 patients with rdnc aus3-type infections , and identical isolates were recovered from each fish tank .
four matched sets of isolates , 2 from 2000 and 2 from 2003 , were further examined ( table ) .
one isolate ( src50 ) characterized previously ( 7 ) was used as a control ( table ) . *
all isolates were resistant to ampicillin , chloramphenicol , streptomycin , spectinomycin , sulfonamides , and tetracycline .
rdnc aus2 and rdnc aus3 are 1 var and 3b var phage - typing variants , respectively , and are identifiable and reproducible phage - typing patterns awaiting formal assignment by the world health organization designated international reference laboratory , colindale , uk .
sgi1 , salmonella genomic island 1 ; nd , strain not available for molecular analysis .
to determine if the resistance phenotype of these strains was due to sgi1 ( 4,5,7,8,11 ) , polymerase chain reaction ( pcr ) with primer pairs shown in figure 1 was used as previously described ( 7 ) .
the left and right junctions of sgi1 with the chromosome and of in104 with sgi1 were present in all cases .
regions containing the gene cassettes were amplified by using standard primers ( l1 and r1 ) in the 5- and 3-conserved segments of class 1 integrons .
fragments of 1.0 and 1.2 kb were amplified from all isolates , and digestion of these amplicons with rsai generated a profile ( data not shown ) that was indistinguishable from the pattern for the 2 amplicons containing the aada2 and blap1 cassettes found in in104 and s. paratyphi b dt+ isolates src49 and src50 from 2001 ( 7 ) .
the aada2 gene cassette was linked to so26 in the sgi1 backbone , which indicates that it is on the left , as in in104 , and the expected 1.8-kb pcr fragment was generated by using primers in groel and blap1 ( figure 1 ) , which places the blap1 cassette on the right .
southern hybridization of xba i digested whole - cell dna with a probe for the flor gene as described previously ( 7 ) identified a band of 12 kb , which is consistent with an sgi1 structure identical to that reported previously ( 7,8,11 ) and the groel - blap1 amplicon linked this 12-kb xbai fragment with the adjacent 4.3-kb xbai fragment ( figure 1 ) . to obtain further evidence for the identity of the matched human and fish tank
isolates , macrorestriction analyses of xbai digested whole - cell dna by pulsed - field gel electrophoresis ( pfge ) were performed as previously described ( 12 ) .
several studies ( 36,13 ) suggest that s. paratyphi b dt+ isolates possess considerable genetic heterogeneity .
the band patterns for all sgi1-containing s. paratyphi b dt+ were identical from humans and fish tanks with phage type rdnc aus3 ( figure 2a ) and aus2 ( data not shown ) .
is200 profiles were also analyzed by hybridization of an is200 probe with pst i digested whole - cell dna as described elsewhere ( 6 ) .
again , all strains showed identical profiles ( figure 2b and data not shown ) that differed by 1 band from profile ip1 recently described ( 6 ) .
thus , matched isolates from humans and their fish tanks were indistinguishable from each other .
pulsed - field gel electrophoresis ( pfge ) and is200 profiles of salmonella enterica serovar paratyphi b dt+ isolates positive for salmonella genomic island 1 .
xba i digested whole - cell dna was separated by pfge as previously described ( 12 ) .
molecular mass markers ( lane m ) are low - range pfge markers ( new england biolabs , beverly , ma , usa ) composed of concatamers of bacteriophage lambda dna .
psti digests of whole - cell dna were separated and hybridized with an is200 digoxigenin ( dig)labeled probe .
molecular mass markers ( lane m ) are dig - labeled bacteriophage lambda dna digested with hindiii ( roche diagnostics , castle hill , new south wales , australia ) .
primers and polymerase chain reaction conditions used to generate the is200 probe have been previously described ( 6 ) .
an unusual observation in this study was that isolates with different phage types showed identical pfge and is200 profiles , indicating that they represented a clonal cluster .
the control strain src50 ( rdnc ) also displayed the same patterns , demonstrating that it also is a member of the same clone .
thus , variation in phage type ( table ) appears to have occurred within a single clone .
variation in phage type has also been reported in other studies of multidrug - resistant s. paratyphi b dt+ strains ( 46 ) , although a number of related but slightly different xbai pfge patterns were observed in those studies .
this finding suggests that all multidrug - resistant s. paratyphi b dt+ found globally have a single origin , but that variations , possibly because of acquisition of other temperate phages or plasmids , have arisen over time .
however , direct comparisons of strains from different countries will be needed to confirm this hypothesis .
this is the first definitive report showing that ornamental fish tanks are a reservoir for multidrug - resistant s. paratyphi b dt+ ( apcmsmspsutc phenotype ) containing sgi1 that causes severe disease in humans , particularly young children .
in addition to containing sgi1 , the matched isolates from humans and their fish tanks had the same phage type and the same xbai macrorestriction digest pattern and is200 profile .
these findings identify home aquariums containing tropical fish as the most important , although not necessarily the only , source of multidrug - resistant s. paratyphi b dt+ .
the fact that 12%14% of australian households have ornamental fish ( 14 ) and as many as 12 million american and 1 million canadian families own domestic aquariums ( 9 ) , together with the young age of most affected patients , indicate that multidrug - resistant s. paratyphi b dt+ in home aquariums is a risk factor for salmonella infection and thus becomes a public health issue . | multidrug - resistant salmonella enterica serovar paratyphi b dt+ isolates from patients with gastroenteritis were identical with isolates from their home aquariums .
matched isolates had identical phage types , xbai and is200 profiles , and salmonella genomic island 1 ( sgi1 ) .
ornamental fish tanks are reservoirs for sgi1-containing s. paratyphi b dt+ . |
theoretical concerns and sporadic evidence have not been able to convince the field to change its dislike for exact replication .
however , this pattern may now change , because far more powerful , and hopefully more convincing , evidence has emerged from the reproducibility project , led by the center for open science in charlottesville , va . in this four - year project11
, 270 experienced investigators joined forces to conduct exact and adequately powered replications of 100 studies that had been published in three leading psychology journals .
the exercise was carried out with exemplary rigour and involved close communication with the original authors to ensure that the replication adhered as faithfully as possible to the original experimental conditions .
there are different statistical approaches to define successful replication , but all of these suggested that nearly two - thirds of the original findings were false - positives , with worse performance in social psychology than in cognitive psychology.11 for example , one replication study tried to replicate whether participants primed with close spatial distances would report stronger feelings of closeness to their family , siblings , and hometown than participants primed with long distances , as proposed in an earlier paper published in 2008.12 despite using identical stimulus materials , dependent variables , and analysis strategies , the replication effort could not replicate the original findings on spatial priming and emotional closeness .
another replication study aimed to replicate that reduced self - regulation resources correlate with increased biases in confirmatory information processing , as previously published.13 the original paper had shown that the depletion of self - regulation resources influences the search and the processing of standpoint consistent information in a personnel decision case , even when confronted with an alternative explanation , i.e. the ego threat , with associated failure cognitions and negative emotions .
more examples and details on the studies replicated can be found in https://osf.io / ezcuj/. the results of the reproducibility project caused a flurry of interest in the scientific community and the general public .
some of the immediate responses were wrong or counter - productive . on one extreme , commenters suggested that psychology is not a science and should be abandoned or be called an art . on the other extreme ,
some dismissed the failures to replicate as having been due presumably to unknown differences between the original experimental setups and the replication attempts .
it is a very important science , and , as the reproducibility project reminds us , has been at the forefront of the study of the scientific method and its biases .
in fact , those that have recently started performing replication experiments have seen very high replication failure rates , even higher than those of the reproducibility project in psychology.17,18 meanwhile disciplines that have adopted replication in large - scale , e.g. , genetic epidemiology , have seen dramatic improvements in the reliability of their results.19 many fields of neuroscience and neurobiology are characterized by the conduct of small , underpowered studies5 and reproducibility is likely to be low .
thus , what we have documented in the reproducibility project may be a pattern that affects many other disciplines .
it is also easy to refute the suggestion that unknown experimental differences are the chief cause of irreproducibility .
if that were the case , one would have seen larger as well as smaller effects in the replication studies , compared to the originals .
in fact , the replication effect sizes were almost always markedly lower in the replication efforts , rendering them statistically non - significant .
probably most of the replication failures in psychological science are due to bias in the original results .
it is not possible to pinpoint exactly which specific study was biased and how bias exactly happened
however , the notion that all results are correct despite failures to reproduce them amounts to irresponsible hand - waving .
if we want a research finding to make any claim to generalizability , or better yet to be used for practical purposes , other scientists should be able to reproduce it relatively easily .
no one would like to fly in a plane that has flown successfully only once , especially if its manufacturers are satisfied that it flew only once and do nt mind that it may crash on its second flight . and
of what use would a plane be if it flew once and was dismantled , and afterwards no one could rebuild it ? in the reproducibility project , one - third of the 147 studies that were identified as possible targets for replication were not picked by any of the 270 replicators , since they were felt to be too difficult , if not impossible , to even try to replicate.11 it is unclear what the value of research is when no one , other than the original scientists , can ever approach it . among the studies in the project for which replications were made eventually , difficulty in building the replication experiments
was a predictor of replication failure.11 the reproducibility project will hopefully lead to a better appreciation of the need for incorporating exact replication more routinely in the life - cycle of research in psychological science .
there are , however , still many unanswered questions and concerns about how to optimally implement a replication agenda .
hastily conceived , suboptimal efforts may even do harm by generating spurious results and confusion .
while this may be seen as eroding a discovery budget that is already constrained , such a perspective would be misleading .
replication is not some sort of unfortunately imposed policing ; it is actually an integral part , perhaps the most integral part , of the scientific discovery process .
if the current situation is such that the majority of discoveries are false , then replication is the most essential element in any true discovery .
replications also allow us to identify rapidly the avenues of research that warrant further investigation and have the best potential for future yield . in short , more reliance on replication can help save us from fund - wasting dead - ends and false - positives .
some other scientific fields have accepted this as a norm . in genetic epidemiology , for example , it is impossible to publish anything in a high - profile journal without independent replication .
however , in the field of psychology there may be insurmountable barriers to the adoption of this principle .
these include practical difficulties ( as discussed above , e.g. for very complex experiments ) .
it may be necessary to target replication efforts in a more limited , strategic way .
unchallenged.20 unchallenged research would have to be treated with extra caution as more likely false than true , perhaps with substantial variability across sub - disciplines .
samples of studies of different types , and stemming from different sub - disciplines , would be subjected to replication periodically to examine what is the current replication performance of the sub - discipline .
therefore one would know that working in field x with study design y carries a z percent risk of non - replication .
such figures would change over time , particularly if the field were to adopt more safeguards to improve its overall research practices .
these safeguards could include registration of research protocols prior to experiment , data sharing , team science approaches or other practices that improve transparency , efficiency , and reliability.2123 for the more influential and heavily cited studies , the imperative for independent exact replication should be very hard to resist , these studies should be subjected to replication .
it would make little sense to neglect to replicate a study upon which hundreds or thousands of other investigations depend .
finally , studies that aim to inform practical applications or otherwise affect humans , such as treatments for psychological problems , should have thorough replication as a sine qua non , before being adopted in everyday practice . at a first stage , such a replication science agenda is also likely to require a very small amount of funds , perhaps 3 to 5 percent of the current research budget a bargain if it reduces the 50 to 90 percent of the research budget that currently seems to be wasted on irreproducible research .
that said , the devil can be in the details , such as who will fund replications , when should they take place , and how should they be conducted .
editors and reviewers also need to become friendly to good replications24,25 ; publishing replications will only greatly encourage replication . to get where we need to go , all action plans will need to have strong grass - roots endorsement by the scientific community .
the reproducibility project , and the favorable responses to it , show that many scientists care deeply about making research more reproducible .
there is no reason to doubt that the general public would also want the same . | editor s note : science has always relied on reproducibility to build confidence in experimental results .
now , the most comprehensive investigation ever done about the rate and predictors of reproducibility in social and cognitive sciences has found that regardless of the analytic method or criteria used , fewer than half of the original findings were successfully replicated . while a failure to reproduce does not necessarily mean the original report was incorrect
, the results suggest that more rigorous methods are long overdue . |
periodontium refers to the tissues that collectively invest and support the teeth and consists of the gingiva , periodontal ligament , alveolar bone , and cementum .
the structure and composition of the periodontium are affected in many acquired and heritable diseases , and the most significant among these is periodontal disease .
the hallmarks of periodontal disease are destruction of soft connective tissues , bone loss , and loss of connective tissue attachment to the cementum . these alterations , if left untreated , lead to tooth loss .
periodontal disease is a complex infectious disease resulting from the interplay of bacterial infection and host response to bacterial challenge , and the disease is modified by environmental , acquired risk factors and genetic susceptibility and is defined as an inflammatory disease of supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms , the key organisms including aggregatibacter actinomycetemcomitans , porphyromonas gingivalis , prevotella intermedia , tannerella forsythia , fusobacterium nucleatum , peptostreptococcus micros , and campylobacter rectus .
the aim of periodontal therapy is to regenerate and restore the various periodontal components affected by disease to their original form , function , and consistency .
for decades , periodontists have sought ways to repair the damage that occurs during periodontitis .
this has included the use of a range of surgical procedures , the use of a variety of grafting materials ( autologous bone and bone marrow , allograft , xenografts , and various manmade bone substitutes ) and growth factors , and the use of barrier membranes .
although these techniques had limited success , the need for a more effective regenerative approach resulted in the development of procedures that use biological mediators and tissue - engineering techniques .
the principal requirements for tissue engineering are the incorporation of appropriate numbers of responsive progenitor cells and the presence of bioactive levels of regulatory signals within an appropriate extracellular matrix or carrier construct .
recent advances in mesenchymal stem cell isolation , growth factor biology , and biodegradable polymer constructs have set the stage for successful tissue engineering of many tissues , of which the periodontium could be considered a prime candidate for such procedures .
stem cells are unspecialized cells that develop into the specialized cells that make up different types of tissue in the human body . they are vital to the development , growth , maintenance , and repair of our brain , bones , muscles , nerves , blood , skin , hair , and other organs .
the isolation , culture , and partial characterization of stem cells isolated from human embryos were reported in the november of 1998 .
stem cell therapy is a treatment that uses stem cells , or cells that come from stem cells , to replace or to repair a patient 's cells or tissues that are damaged and stem cell technologies have , or are anticipated to have , applications for basic science ( study of complex processes ) , medical research ( to produce large numbers of genetically uniform cultures of organ tissues ; e.g. , liver , muscle , or neural ) , and therapies ( repair or replace damaged or diseased tissues ) .
all stem cells , no matter what their source , are unspecialized cells that give rise to more specialized cells .
they serve as the body 's repair system by renewing themselves and replenishing more specialized cells in the body , and the easiest way to categorize stem cells is by dividing them into two types : mature and early .
mature stem cells are found in specific mature body tissues as well as the umbilical cord and placenta after birth .
early stem cells , often called embryonic stem cells , are found in the inner cell mass of a blastocyst after approximately 5 days of development .
stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture .
highly plastic adult stem cells from a variety of sources , including umbilical cord blood and bone marrow , are routinely used in medical therapies .
embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies .
potency specifies the differentiation potential ( the potential to differentiate into different cell types ) of the stem cell , such as totipotent , pluripotent , multipotent , oligopotent , and unipotent . in order for successful periodontal regeneration to occur , it will be necessary to use and recruit progenitor cells that can differentiate into specialized cells with a regenerative capacity , followed by proliferation of these cells and synthesis of the specialized connective tissues that they are attempting to repair . clearly , a tissue - engineering approach for periodontal regeneration will need to utilize the regenerative capacity of cells residing within the periodontium , and would involve the isolation of such cells and their subsequent proliferation within a three - dimensional ( 3d ) framework with implantation into the defect . in wound healing , the natural healing process usually results in tissue scarring or repair . using tissue engineering , the wound - healing process
this manipulation usually involves one or more of the following three key elements : the signaling molecules , scaffold or supporting matrices , and cells ; the idiosyncratic feature and characteristic of the three key elements are illustrated in tables 13 .
characteristics of signaling molecules in periodontal tissue engineering characteristics of scaffold / supporting matrices in periodontal tissue engineering characteristics of stem cells in periodontal tissue engineering the various challenges that exist in periodontal regeneration are illustrated below :
a major unmet challenge is the modulation of the exuberant host response to microbial contamination that plagues the periodontal wound .
dual delivery of host modifiers and anti - infective agents is probably necessary to optimize periodontal regeneration.the necessary interactions of multiple cell lineages should be clarified .
these include cementogenic cells , fibroblasts , and osteogenic cells.despite the important role of exogenously delivered cells in the regeneration of severe periodontal defects , it is advantageous to attract endogenous periodontal tissue - forming cells by growth and/or trophic factors .
a major unmet challenge is the modulation of the exuberant host response to microbial contamination that plagues the periodontal wound .
dual delivery of host modifiers and anti - infective agents is probably necessary to optimize periodontal regeneration .
these include cementogenic cells , fibroblasts , and osteogenic cells . despite the important role of exogenously delivered cells in the regeneration of severe periodontal defects , it is advantageous to attract endogenous periodontal tissue - forming cells by growth and/or trophic factors . as
human stem cell research is a relatively new area , companies developing cell therapies face several types of risk , and some are not able to manage them thus becoming highly speculative enterprises .
present clinical trials are being performed on recombinant human fibroblast growth factor-2 , human platelet - derived growth factor , and tricalcium phosphate ( gem-21 ) .
looking at the ongoing clinical trials , it is too early to tell whether all therapies based on stem cells will prove to be clinically effective .
thus , despite extensive research , there still remain problems with stem cell therapy because , in many cases , deep and exhaustive studies to determine the exact biology of stem cells are omitted and there are increasing pressures to start with insufficiently controlled clinical trials .
, scientists have discovered a wide array of stem cells that have unique capabilities to self - renew , grow indefinitely , and differentiate or develop into multiple types of cells and tissues . coating the surface of every cell in the body
are specialized proteins , called receptors , which have the capability of selectively binding or adhering to other signaling molecules .
there are many different types of receptors that differ in their structure and affinity for the signaling molecules .
normally , cells use these receptors and the molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body .
the importance of this new technique is that it allows the tracking of stem cells as they differentiate or become specialized .
scientists have inserted into a stem cell a reporter gene called green fluorescent protein or gfp .
these discovery tools are commonly used in research laboratories and clinics today , and will probably play important roles in advancing stem cell research .
one of them is that a single marker identifying pluripotent stem cells , those stem cells that can make any other cell , has yet to be found . as new types of stem cells
are identified and their research applications become increasingly complex , more sophisticated tools will be developed to meet investigators needs . for the foreseeable future , markers will continue to play a major role in the rapidly evolving world of stem cell biology .
stem cells are unspecialized cells that develop into the specialized cells that make up different types of tissue in the human body . they are vital to the development , growth , maintenance , and repair of our brain , bones , muscles , nerves , blood , skin , hair , and other organs .
the isolation , culture , and partial characterization of stem cells isolated from human embryos were reported in the november of 1998 .
stem cell therapy is a treatment that uses stem cells , or cells that come from stem cells , to replace or to repair a patient 's cells or tissues that are damaged and stem cell technologies have , or are anticipated to have , applications for basic science ( study of complex processes ) , medical research ( to produce large numbers of genetically uniform cultures of organ tissues ; e.g. , liver , muscle , or neural ) , and therapies ( repair or replace damaged or diseased tissues ) .
all stem cells , no matter what their source , are unspecialized cells that give rise to more specialized cells .
they serve as the body 's repair system by renewing themselves and replenishing more specialized cells in the body , and the easiest way to categorize stem cells is by dividing them into two types : mature and early .
mature stem cells are found in specific mature body tissues as well as the umbilical cord and placenta after birth .
early stem cells , often called embryonic stem cells , are found in the inner cell mass of a blastocyst after approximately 5 days of development .
stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture .
highly plastic adult stem cells from a variety of sources , including umbilical cord blood and bone marrow , are routinely used in medical therapies .
embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies .
potency specifies the differentiation potential ( the potential to differentiate into different cell types ) of the stem cell , such as totipotent , pluripotent , multipotent , oligopotent , and unipotent .
in order for successful periodontal regeneration to occur , it will be necessary to use and recruit progenitor cells that can differentiate into specialized cells with a regenerative capacity , followed by proliferation of these cells and synthesis of the specialized connective tissues that they are attempting to repair .
clearly , a tissue - engineering approach for periodontal regeneration will need to utilize the regenerative capacity of cells residing within the periodontium , and would involve the isolation of such cells and their subsequent proliferation within a three - dimensional ( 3d ) framework with implantation into the defect . in wound healing , the natural healing process usually results in tissue scarring or repair . using tissue engineering ,
this manipulation usually involves one or more of the following three key elements : the signaling molecules , scaffold or supporting matrices , and cells ; the idiosyncratic feature and characteristic of the three key elements are illustrated in tables 13 .
characteristics of signaling molecules in periodontal tissue engineering characteristics of scaffold / supporting matrices in periodontal tissue engineering characteristics of stem cells in periodontal tissue engineering
the various challenges that exist in periodontal regeneration are illustrated below :
a major unmet challenge is the modulation of the exuberant host response to microbial contamination that plagues the periodontal wound .
dual delivery of host modifiers and anti - infective agents is probably necessary to optimize periodontal regeneration.the necessary interactions of multiple cell lineages should be clarified .
these include cementogenic cells , fibroblasts , and osteogenic cells.despite the important role of exogenously delivered cells in the regeneration of severe periodontal defects , it is advantageous to attract endogenous periodontal tissue - forming cells by growth and/or trophic factors .
a major unmet challenge is the modulation of the exuberant host response to microbial contamination that plagues the periodontal wound .
dual delivery of host modifiers and anti - infective agents is probably necessary to optimize periodontal regeneration .
these include cementogenic cells , fibroblasts , and osteogenic cells . despite the important role of
exogenously delivered cells in the regeneration of severe periodontal defects , it is advantageous to attract endogenous periodontal tissue - forming cells by growth and/or trophic factors .
as human stem cell research is a relatively new area , companies developing cell therapies face several types of risk , and some are not able to manage them thus becoming highly speculative enterprises .
present clinical trials are being performed on recombinant human fibroblast growth factor-2 , human platelet - derived growth factor , and tricalcium phosphate ( gem-21 ) .
looking at the ongoing clinical trials , it is too early to tell whether all therapies based on stem cells will prove to be clinically effective .
thus , despite extensive research , there still remain problems with stem cell therapy because , in many cases , deep and exhaustive studies to determine the exact biology of stem cells are omitted and there are increasing pressures to start with insufficiently controlled clinical trials .
in recent years , scientists have discovered a wide array of stem cells that have unique capabilities to self - renew , grow indefinitely , and differentiate or develop into multiple types of cells and tissues .
coating the surface of every cell in the body are specialized proteins , called receptors , which have the capability of selectively binding or adhering to other signaling molecules .
there are many different types of receptors that differ in their structure and affinity for the signaling molecules .
normally , cells use these receptors and the molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body .
the importance of this new technique is that it allows the tracking of stem cells as they differentiate or become specialized .
scientists have inserted into a stem cell a reporter gene called green fluorescent protein or gfp .
these discovery tools are commonly used in research laboratories and clinics today , and will probably play important roles in advancing stem cell research .
there are , however , limitations to this research . one of them is that a single marker identifying pluripotent stem cells , those stem cells that can make any other cell , has yet to be found . as new types of stem cells
are identified and their research applications become increasingly complex , more sophisticated tools will be developed to meet investigators needs . for the foreseeable future , markers will continue to play a major role in the rapidly evolving world of stem cell biology .
regeneration of a functional and living tooth is one of the most promising therapeutic strategies for the replacement of a diseased or damaged tooth.[810 ] recent advances in dental stem cell biotechnology and cell - mediated murine tooth regeneration have encouraged researchers to explore the potential for regenerating living teeth with appropriate functional properties.[1113 ] murine teeth can be regenerated using many different stem cells to collaboratively form dental structures in vivo . in addition , dentin / pulp tissue and cementum / periodontal complex have been regenerated by human dental pulp stem cells and periodontal ligament stem cells , respectively . however , owing to the complexity of human tooth growth and development , the regeneration of a whole tooth structure , including enamel , dentin / pulp complex , and periodontal tissues , as a functional entity in humans is not possible given the available regenerative biotechnologies .
the end goal of tissue engineering is to develop products capable of healing diseased or lost tissues and organs ; thus , representing a departure from conventional biomedical research , whose primary focus is an understanding of mechanisms .
this does not imply that the understanding of mechanisms is unimportant in tissue engineering . instead , an understanding of the mechanisms of interactions among cells , growth factors , and biomaterials undoubtedly will advance the end goal of developing cell - based therapies and off - the - shelf tissue - engineering products ; conversely , craniofacial tissue engineering could not have advanced to the current stage without the incorporation of interdisciplinary skill sets of stem cell biology , bioengineering , polymer chemistry , mechanical engineering , robotics , etc .
thus , craniofacial tissue engineering and regenerative dental medicine are integral components of regenerative medicine . despite the accumulation of molecular information and our understanding of the regulation of tooth development ,
perhaps , one day , we will be able to isolate cells that have the capacity to form teeth and then tooth development could be initiated in vitro .
after initiation , the tooth germ could either be transplanted into the mouth or it could be cultured in vitro
. this approach would be the most difficult as it would require a thorough knowledge of all processes that govern the formation of the proper three - dimensional structure of the tooth .
alternatively , it is possible that tooth development could be initiated in vivo by applying specific growth and differentiation factors . in summary , these are still early days of periodontal tissue regeneration and more recent developments in basic science indicate that these approaches are unquestionably practical and , given their promise , worth exploring . | the structure and composition of the periodontium are affected in many acquired and heritable diseases , and the most significant among these is periodontal disease .
periodontal regeneration is considered to be organically promising but clinically capricious .
the principal requirements for tissue engineering are the incorporation of appropriate numbers of responsive progenitor cells and the presence of bioactive levels of regulatory signals within an appropriate extracellular matrix or carrier construct . stem cell therapy is a treatment that uses stem cells , or cells that come from stem cells , to replace or to repair a patient 's cells or tissues that are damaged . and , recent progress in stem cell research and in tissue engineering promises novel prospects for tissue regeneration in dental practice in the future , with regeneration of a functional and living tooth as one of the most promising therapeutic strategies for the replacement of a diseased or damaged tooth . |
catamenial pneumothorax ( cp ) is a spontaneous recurrent pneumothorax occurring in women in reproductive age .
the etiology of cp has been associated with thoracic endometriosis and is its most common presentation .
a case of right catamenial pneumothorax in a 38 year old woman is presented in which three episodes of cp occurred within 72 h of menses in a 6 month period .
after surgical pleurodesis and based on final pathology of resected lesion , hormonal treatment was started .
catamenial pneumothorax ( cp ) is a rare clinical entity characterized by lung collapse during menstruation , believed to be secondary to pleural endometriosis .
nearly all catamenial pneumothorax occur on the right side as pleural lesions are almost exclusively right - sided .
diagnostic imaging is based on high resolution computed tomography ( hrct ) and , preferably , magnetic resonance imaging ( mri ) since it is able to detect the blood products in the endometrial deposits .
however the lack of macroscopic findings at surgery makes this condition still under - diagnosed .
based on the solitary diaphragmatic localization of endometriosis in our case we preferred to limit surgery to videothoracoscopic pleurodesis and start hormonal treatment with successful outcome .
catamenial pneumothorax is the most common presentation of thoracic endometriosis syndrome and should always be suspected in women in childbearing age .
treatment option are still debated but best results are achieved by videothoracoscopic pleurodesis combined with hormonal therapy .
catamenial pneumothorax ( cp ) is a spontaneous recurrent pneumothorax occurring in women in reproductive age .
the etiology of cp has been associated with thoracic endometriosis namely the presence of endometrial - like tissue in the thoracic cavity .
thoracic endometriosis is considered a rare condition , usually underdiagnosed , which consists of 4 different clinical entities : catamenial pneumothorax , catamenial hemothorax , hemoptysis and pulmonary nodules . about 60% of pulmonary endometriosis cases
etiopathogenesis of thoracic endometriosis is not clear and the most accredited theory explains the retrograde implantation of endometrial tissue by lymphatic and haematogenous dissemination or presence of diaphragmatic defects .
cp is the most common presentation of thoracic endometriosis syndrome ( tes ) , with a reported incidence of 2.85.6% .
cp accounts for > 30% of all cases of spontaneous pneumothorax in young women with a peak incidence between 30 and 35 years .
cp typically occurs within 24 h before and 72 h after the onset of menses and appears almost exclusively in the right hemithorax .
a combined surgical and hormonal treatment is considered the best management of cp , as postoperative recurrences are common and hormonal therapy is useful to prevent them .
we present a case of recurrent catamenial pneumothorax due to a single diaphragmatic endometriosis localization which was successfully treated with combined surgical and hormonal therapy .
a 38-year - old otherwise healthy woman had 3 episodes of recurrent spontaneous right pneumothorax in the past six months .
two months later the patient was hospitalized because of shortness of breath and chest pain and a 2nd right pneumothorax was diagnosed which also resolved spontaneously after 3 weeks .
after two more months a 3rd episode happened always on the right side , and a better analyzed clinical history revealed that all episodes were related to peri - menstruation period with the 3rd one occurring during menses .
all episodes of pneumothorax were evaluated by chest roentgenogram and computed tomography ( ct ) which did not show any blebs or bullae .
although the patient did not have any history of pelvic or abdominal pain , her serum ca125 level was normal ( 22 g / ml ) , and further evaluation by pelvic ultrasonography and magnetic resonance imaging ( mri ) excluded the presence of pelvic endometriosis , the hypothesis of catamenial pneumothorax was taken into account .
the patient underwent a videothoracoscopic exploration of the right pleural cavity under general anesthesia and single - lung ventilation by a first posterolateral access .
the examination of pleural cavity showed a single typical purple red spot on the right posterior pleural diaphragmatic surface strongly suggestive of endometriosis ( fig .
a second incision was made to perform pleurodesis by mechanical abrasion and surgical resection of diaphragmatic lesion .
after control of hemostasis a 24 ch drain was placed in pleural space and full lung re - expansion achieved .
the chest tube was left in place until postoperative day 3 when the patient was discharged .
final pathology confirmed an endometriosis - related catamenial pneumothorax ( er cp ) according the most updated classification by legras et al .
soon after surgery the patient started a medical treatment with 17-etinil testosterone 800 mg daily which was shifted to dienogest 2 mg daily due to marked untolerable hypoestrogenic side effects following gynecologist s outpatient follow up . at 6 months from surgery no recurrence has been observed .
catamenial pneumothorax ( cp ) is a rare clinical entity characterized by lung collapse during menstruation ( catamenial is a derivation of the greek term katamenios that refers to monthly ) , believed to be secondary to endometriosis within the pleural cavity and causing 1/3 of spontaneous pneumothorax in women of child - bearing age .
although the progressive improvement in imaging techniques and minimally invasive procedures , the lack of macroscopic findings at surgery makes this condition still under - diagnosed with a reported incidence of 35% .
the disease more frequently occurs in premenopausal women aged 3050 who have a history of infertility , severe endometriosis and recurrent spontaneous pneumothorax within 72 h of the onset of menses .
pleural endometriosis presents as catamenial pneumothorax , catamenial hemothorax , catamenial pneumomediastinum and chest pain .
the metastatic theory assumes the transdiaphragmatic passage of endometrial tissue by lympho - hematogenous dissemination or congenital fenestration .
the anatomical theory calls for dissolution of cervical mucus plug during the menses with migration of cells through vagina , uterus , fallopian tubes to peritoneum and air reaching the chest by diaphragmatic congenital fenestrations . in the hormonal theory
congenital diaphragmatic defects are more common on the right side and this can explain the higher prevalence of right - sided cp .
the prevalence of diaphragmatic fenestrations varies from 23% to 88% , and diaphragmatic fenestrations can be associated with endometrial implants .
the diaphragmatic defect(s ) can be single or multiple , usually located at the central tendon , and are described as perforations , holes , fenestrations , pores , porosities , and stomata .
holes proven only by diagnostic pneumoperitoneum , tiny holes ( 13 mm ) , or larger defects ( > 10 mm ) . as seen in this case and in most reports , nearly in all cases catamenial pneumothorax occurs on the right side as pleural lesions are almost exclusively right - sided .
this predilection is probably due to the more extensive diaphragmatic lymphatic drainage on the right side and the clockwise peritoneal circulation that sweeps endometrial implants to the right diaphragm .
diagnostic imaging is based on high resolution computed tomography ( hrct ) to detect endometrial deposits in the lung and pleura and pelvic ultrasound due to coexistence of pulmonary and pelvic endometriosis in 5080% of cases .
both thoracic and pelvic mri are considered superior to ct due to the blood products in the endometrial deposits .
typical mri pattern shows a centrally or peripherally located low - intensity area in the hyperintense cyst on t2 weighted images ( shading ) .
thoracic mri lesions are hyperintense on t2-weighted spin - echo , increase in size at the time of menstruation and show pronounced uptake contrast agent as compared with the intermenstrual period .
ca125 ( cancer antigen 125 ) is a tumor marker that is widely used to monitor ovarian cancer because it is overexpressed in ovarian cancer cells and secreted into the blood .
ca-125 levels may be elevated in a variety of benign ovarian conditions , such as pelvic inflammatory disease , endometriosis , and benign neoplasms .
endometriosis has also been associated with increased levels of ca-125 , and although it is not considered a specific marker , it can assist in early diagnosis of endometriosis - related pneumothorax .
suggest ca-125 measurement be included in females of reproductive age presenting with primary spontaneous pneumothorax without gynaecological orientation and without clinical signs of malignant and nonmalignant conditions that injure mesothelial cells .
they conclude that this biological marker may favor indication of videothoracoscopy and hormonal therapy at an early stage in the prevention of catamenial pneumothorax recurrences , and should be evaluated prospectively in a larger - scale study .
moreover in a study conducted by pickhardt and hanson to determine the prevalence , work - up , and outcomes of indeterminate adnexal masses in asymptomatic women , the only case of elevated ca-125 was associated with endometriosis .
however , ca-125 levels only reflect the amount of endometrial tissue , and it is currently believed that the usefulness of such measurements is low . as a matter of fact in our case ca-125 levels
medical treatment using 17-etinil testosterone a derivative of the synthetic steroid ethisterone has been advised since it suppresses the production of gonadotropins , thus leading to a condition of pseudomenopause and finally a remission of the disease
. the patient will be hypoestrogenic due to suppression of the ovarian steroidogenesis . a synthetic oral progestin having progestational and antigonadotrophic effects , but no androgenic , glucocorticoid or mineralocorticoid activity is dienogest . it is believed to induce both an antiproliferative effect on endometriotic cells and a reduction of estrogen levels due to the inhibition of ovarian function .
a regimen with 2 mg a day is considered as the optimal dose for provision of efficacy and safety in endometriosis studies . in fact
this dosage moderately suppresses estradiol levels , and has high oral bioavailability and a half - life suitable for once - daily administration .
compared to gnrh analogs dienogest seems to cause less hypoestrogenic side effects and little changes in bone markers and bone mineral density offering better advantages in safety and tolerability .
however because of endometriosis is an hormone dependent disease , when the medical treatment is stopped there is a relapse of the disease and hormonal therapy alone is associated with a recurrence rate exceeding 50% within 6 months after treatment is stopped .
the surgical approach has been suggested to be superior to hormonal therapy alone since it can address most of the intrathoracic pathologic features .
the association of surgical treatment followed by approximately 6 months of hormone therapy may achieve up to 45 month follow up without recurrence and is presently the preferred approach for treatment and prevention .
surgical treatment should be timed during menstruation for optimal visualization of pleurodiaphragmatic endometriosis as we did in our case .
for such a purpose videothoracoscopy is a very useful tool since it assesses both the diaphragmatic surface and the pleural cavity and is able to detect typical blueberry - like spots or purple red foci , although it has been reported that in 8.5% of explored cases no pathologic findings were demonstrated . .
suturing the defect , endoscopic resection of the area , covering the diaphragmatic surface with polyglactin mesh are among the commonly reported surgical procedures which , however , do not prevent the risk of cp to recur .
therefore , in patients with endometriosis - related catamenial pneumothorax , pleurodesis plays a major role , when microscopic endometrial foci , or newly implanted lesions can not be detected . in our case
we found a single typical purple red spot on the right posterior diaphragmatic surface but no other deposits nor fenestrations were detected .
this is why we preferred to limit our surgical procedure to resection of the lesion and mechanical pleurodesis , the only successful reported procedure , and combine it with the hormonal treatment to achieve the best outcome .
catamenial pneumothorax is the most common presentation of thoracic endometriosis syndrome and should always be suspected in women with a suggestive history of recurrent pneumothorax and pelvic / abdominal endometriosis .
although the number of reported cases with catamenial pneumothorax is limited , conservative treatment is insufficient . videothoracoscopy provides magnification and better visualization of the pleural cavity and diaphragm . among surgical options
videothoracoscopic resection of detected lesions combined with pleurodesis is the most successful one and , associated with hormonal therapy , reduces the risk of recurrence . | highlightscatamenial pneumothorax is related to thoracic endometriosis syndrome.diagnosis of catamenial pneumothorax is a difficult challenge and its treatment is controversial.we report a case of catamenial pneumothorax caused by a single diaphragmatic localization of thoracic endometriosis.successful treatment consisted of combined video - assisted pleurodesis and postoperative hormonal therapy . |
although regional anesthesia offers advantages for intraoperative and postoperative pain relief , it is not without complications.1,2 the majority of complications associated with peripheral nerve blocks are due to local anesthetic toxicity after inadvertent intravascular injection.3 however , there are many other well known complications
. the increasing use of ultrasound guidance for , eg , intraneural or intravascular injections , may reduce some of these risks , although not completely eliminate them .
postoperative management of peripheral nerve blocks , and cohesive standardized steps to prevent complications have received little attention . a case of a significant burn injury after splint placement
it is our hope that this case alerts physicians and others involved in the management of postoperative patients to the challenges of managing a blocked extremity after thermal cast placement .
a 62-year - old woman weighing 61 kg presented for an elective modified right mcbride bunionectomy , right first metatarsal osteotomy , and right second hammertoe correction .
she previously underwent the same surgery for a left hallux valgus deformity , and did very well without complications under regional and general anesthesia .
preoperatively , at approximately 11:40 am on the day of surgery , popliteal and saphenous nerve blocks were administered for postoperative analgesia .
the skin was anesthetized with 5 ml of 1% lidocaine . under ultrasound guidance , a 22-gauge 3.5-inch spinal needle was inserted , and 40 ml of 0.25% bupivacaine with 1:200,000 epinephrine was injected incrementally , with aspiration every 5 ml , 30 ml in the popliteal region , and 10 ml in the saphenous region .
the surgery went as planned , with no complications , and the total tourniquet time was approximately 120 minutes . at the conclusion of surgery at approximately 2:30 pm ,
a soft padded bulky jones dressing was placed over the lower extremity with a plaster posterior splint ( bsn medical specialist ) .
the temperature of the water was noted to be warm , but no exact temperature was recorded . the splint was folded over slightly at the edge of the splint near the posterior knee .
her stay in the postanesthesia care unit was uneventful until approximately 3:30 pm , when she complained of 6/10 pain in her foot .
it was noted in the nurse s notes that she had difficulty localizing the pain and was given hydromorphone 0.4 mg .
when this did not relieve her pain , the anesthesiologist was called , and it was decided to touch up her block .
at 1605 , popliteal nerve and saphenous nerve blocks were performed using the aforementioned technique , with 20 ml and 10 ml of 0.25% , respectively .
she developed adequate analgesia and was discharged from the postanesthesia care unit at 5:24 pm with 0/10 pain .
she presented to the orthopedic clinic on postoperative day 3 with severe pain in the lower extremity .
the splint and dressing were removed to reveal a partial third degree burn on the posterior lower leg ( figure 1 ) which eventually required a skin graft ( figure 2 ) .
follow - up with the patient after her recovery from the second surgery revealed no further problems or long - term pain conditions .
it decreases postoperative mortality caused by thromboembolic disease and myocardial infarction , improves postoperative analgesia , decreases the incidence of early postoperative cognitive dysfunction , and allows shorter recovery times.1,2 however , there are well known complications , including local anesthetic toxicity , infection , hematoma , nerve damage , and , depending on the area blocked , pneumothorax , hemothorax , and respiratory failure.5 certain situations and surgeries bring additional challenges to regional anesthesia , such as thermal cast placement applied to a blocked extremity .
cast application techniques are known to prevent burn injuries4,6 but , as with any procedure , mistakes still occur .
internal temperatures can reach > 50c with a fast curing plaster cast , and it has been found that > 49c is enough to cause significant burn injuries.6 unfortunately , the pain of a burn injury from a thermal cast can be masked in a patient with a blocked extremity .
peripheral nerve blocks for orthopedic procedures are now becoming commonplace , so increasing awareness of this possible complication to prevent such injuries is needed . for patient safety ,
our institution focuses on leg elevation , moving noninjured digits of the affected limb , and monitoring pulse oximetry to test perfusion of the extremity , but little temperature related .
one study measured the internal temperature using a simple temperature sensor ( temp sensor thermistor 400 series ; tyco healthcare , harrisburg , pa ) and found it to be adequate for evaluating the skin under plaster cast burns.7 this may be a simple technique to evaluate a blocked extremity with a cast .
subjective evaluation of the temperature of the cast by touching or measuring the external temperature is open to individual interpretation , and could introduce too much variability and inaccuracy based on the layering present and type of cast used .
any complaint of pain should be thoroughly investigated by the nursing staff in the postoperative anesthesia care unit , with special consideration given to avoiding thermal injuries , and temperature measurements of the skin should be done prior to the administration of opioids .
the anesthesiologist should be notified early about pain in these patients , should participate in the evaluation , and be mindful of the possibility of thermal burns occurring in the setting of postoperative regional blockade .
it may be necessary to alert the surgeon , and strong consideration should be given to possible removal of the plaster material to assess for thermal injury .
this case is illustrative of the fact that , even in the setting of regional blockade , there may be other reasons for what could be perceived as a failed regional blockade , and additional opioids and/or touch up nerve blocks should not be done without a thorough evaluation of the plaster cast and underlying skin .
while this case involves a competent , awake adult female , it does prompt consideration of the possibility that other patient populations may be at greater risk for this complication due to inability to provide appropriate feedback and communicate possible breakthrough thermal - mediated pain .
these patient populations include children and the elderly , who may not be able to communicate , and patients who have been heavily sedated .
the risk - benefit of performing regional blockade should be assessed prior to undertaking this procedure .
regarding the casting material used , in this case the bsn medical specialist synthetic splint ( bsn medical distribution ltd , brierfield , uk ) , there are generalized precautions that include the warning to the patient that the splint may feel warm
. there are no published precautions regarding the use of this material in conjunction with peripheral nerve blocks.8
it is not known with certainty if the cast used in our patient was too hot , too thick , or if it was adequately padded .
the patient certainly complained of increased pain in the postanesthesia care unit which resulted in an additional block leading to this poor outcome .
there were no indicators that the patient failed to receive adequate blockade , so this breakthrough pain was likely related to unrecognized ongoing thermal injury .
communication with nursing staff , as well as other anesthesiology colleagues , is important when a cast is placed on a blocked extremity to alert those involved in caring for the patient with a splint or cast of the possibility of thermal injury .
the best method to assess for possible thermal injury is not known , but certainly the evaluation should begin early , and involve both the anesthesiologist and the surgeon . this could save patients from the debilitating complication of a burn injury , and improve our postoperative strategies . | although regional anesthesia offers advantages for intraoperative and postoperative pain relief , it is not possible without complications . a case of a significant burn injury after splint placement
is described after a peripheral nerve block was performed for postoperative pain management .
it is our hope that this case alerts physicians and others involved in the management of postoperative patients to the challenges of managing a blocked extremity after thermal cast placement and offers solutions that can be standardized . |
neuromyelitis optica ( nmo ) is a complex severe demyelinating disorder of central nervous system that was primarily considered to be characterized by optic neuritis ( on ) and myelitis .
nmo was considered a disease without brain involvement , which was part of major diagnostic criteria proposed by wingerchuk et al . in 1999 .
ever since the discovery of nmo - igg / anti - aquaporin-4 ( aqp-4 ) antibody in a proportion of patients with clinically proven nmo and in patients with recurrent , isolated , longitudinally extensive spinal cord lesions ( lecs ) or on as well as patients with lecs or on associated with systemic autoimmune disease or with brain lesions , it has resulted in the identification of a spectrum of disorders called nmo spectrum disorders ( nmosd ) .
although positivity of aqp-4 antibody and presence of brain lesions have been introduced in the revised nmo criteria , the presence of on and lecs is still mandatory [ table 1 ] .
in addition , a separate spectrum for aqp-4 antibody positive patients not fitting into the nmo clinical criteria has been proposed [ table 2 ] . revised criteria for the diagnosis of neuromyelitis optica neuromyelitis optica spectrum isolated and asymptomatic brain findings are now increasingly identified at presentation in these cases ; hence , it is imperative to be aware of these brain lesions which might be specific enough to prompt correct diagnosis .
in addition , as the symptoms may be dramatic and may not be neurological , we need to be aware of this entity .
we herein present a case of an adult woman who had a past history of on and presented with recent onset hiccups and intractable vomiting .
a 45-year - old woman came to the emergency department of our hospital with a history of intractable vomiting and hiccups for 2 days . she was admitted and extensively evaluated in the form of blood investigations and endoscopy .
on enquiry , she gave a vague history of loss of vision in the left eye which improved gradually after taking some medication .
small hyperintense specks were seen symmetrically in axial fluid attenuation inversion recovery images in the dorsal aspect of the upper medulla at the caudal end of the fourth ventricle ( area postrema ) [ figure 1 ] .
on sagittal t2-weighted images , thin streak - like hyperintensity was seen [ figure 2 ] .
no restriction of diffusion or definite contrast enhancement was noted [ figures 3 and 4 ] .
fluid attenuation inversion recovery axial image at the level of upper medulla shows tiny hyperintense foci in the dorsal aspect of medulla at the caudal end of fourth ventricle - area postrema ( arrows ) mid sagittal t2-weighted image shows faint hyperintense linear streak in the dorsal aspect of upper medulla near the caudal end of fourth ventricle ( arrow ) diffusion weighted image shows no restriction no enhancement seen on post contrast scan these lesions were so tiny and nondescript that we had missed them on first analysis , however on review , we came to a conclusion that these indeed could be lesions related to nmosd .
the history of vision loss and recovery which could be suggestive of on also proved to be corroborative .
as the patient can not be classified as nmo , at present , due to lack of cord symptoms , she definitely fits the criteria for nmosd .
lack of brain involvement was traditionally one of the major supportive diagnostic criteria ; however , later it was established that asymptomatic brain abnormalities may be seen in up to 60% of the patients with clinically defined nmo .
a number of studies have identified a defined spectrum of brain lesion types in up to 93% of the patients with nmo- and aqp-4 antibody - positive nmosd .
now , with identification of nmosd , as it is increasingly recognized that some patients present with brain symptoms as their first manifestation or develop recurrent brain symptoms without on or myelitis , these brain abnormalities have been called to clinicians and radiologists attention .
nonspecific punctate or small ( 3 mm ) lesions in the deep or , less commonly , subcortical white matter are frequent and usually asymptomatic .
specific lesion types that are helpful in the radiologic diagnosis of nmo / nmosd are as follows :
typical lesions in the peri - ependymal regions surrounding the third ventricle , cerebral aqueduct , and fourth ventricle , wherein the aqp-4 antigen is in abundance
. such lesions within the thalamus and hypothalamus may present with a variety of symptoms such as syndrome of inappropriate antidiuretic hormone secretion , narcolepsy , behavioral change , and hypothermia , hypotension , hypersomnia , and obesity .
brain stem lesions can result in a variety of bulbar symptoms , among which intractable hiccups , nausea , and vomiting are relatively specific for nmo and attributable to the involvement of the area postrema and the nucleus tractus solitariusour patient had similar symptoms attributable to tiny foci of demyelination in area postremaperiependymal / periventricular and corpus callosum lesions situated in proximity to the lateral ventricles , which need to be differentiated from multiple sclerosis ( ms ) plaquesunilateral or bilateral , longitudinally extensive lesions involving the corticospinal tract especially seen in aqp-4 antibody - positive korean populationextensive hemispheric lesions : extensive and confluent hemispheric white matter lesions are tumefactive ( > 3 cm in longest diameter ) or configured as long spindle - like or radial - shaped signal changes following white matter tracts such as the longitudinal fasciculuscortical involvement and leptomeningeal enhancement has been described , but it is very rare .
typical lesions in the peri - ependymal regions surrounding the third ventricle , cerebral aqueduct , and fourth ventricle , wherein the aqp-4 antigen is in abundance .
such lesions within the thalamus and hypothalamus may present with a variety of symptoms such as syndrome of inappropriate antidiuretic hormone secretion , narcolepsy , behavioral change , and hypothermia , hypotension , hypersomnia , and obesity .
brain stem lesions can result in a variety of bulbar symptoms , among which intractable hiccups , nausea , and vomiting are relatively specific for nmo and attributable to the involvement of the area postrema and the nucleus tractus solitarius our patient had similar symptoms attributable to tiny foci of demyelination in area postrema periependymal / periventricular and corpus callosum lesions situated in proximity to the lateral ventricles , which need to be differentiated from multiple sclerosis ( ms ) plaques unilateral or bilateral , longitudinally extensive lesions involving the corticospinal tract especially seen in aqp-4 antibody - positive korean population extensive hemispheric lesions : extensive and confluent hemispheric white matter lesions are tumefactive ( > 3 cm in longest diameter ) or configured as long spindle - like or radial - shaped signal changes following white matter tracts such as the longitudinal fasciculus cortical involvement and leptomeningeal enhancement has been described , but it is very rare .
in addition , these lesions have to be differentiated from ms as in about 5% of the patients with ms may also have the nmo antibody and a subgroup of a nmo patients may be negative for the antibody .
more importantly , some of the treatment options of ms may be useless and may actually worsen nmo . apart from clinical presentations which are usually more fulminant in nmo , initial involvement of cord and optic nerves also point to the diagnosis .
different patterns of spinal cord involvement are noted in the two diseases , long segment cord involvement in nmo ( > 3 segments ) while short segment involvement is seen in ms .
periventricular lesions can be seen in both ms and nmo ; however , typical ms , lesions are discrete , ovoid , perpendicular to the ventricles , and perivenular extended ( e.g. , dawson 's fingers ) , while nmosd lesions are located immediately next to the lateral ventricle , following the ependymal lining in disseminated pattern and are often edematous and heterogeneous . in our case ,
the patient had a past history of on , and in the current episode , presented with non - neurological symptoms .
thus , emphasizing that it is important to know the brain manifestations of nmo / nmosd as these may be the first manifestations of this disease and though many findings may be nonspecific , their characteristic locations and configurations are helpful .
a thorough understanding of diverse brain manifestations is now crucial for early and correct diagnosis of nmosd .
| neuromyelitisoptica ( nmo ) and multiple sclerosis ( ms ) were once considered to be differing manifestation of same auto immune disease , nmo predominantly involving the optic nerve and cord .
now with discovery of nmo antibody the concept has changed and a spectrum of disorders with lesions in brain has been identified . occasionally , brain may be the first or the only site of involvement in these disorders hence it is essential to be aware of this spectrum .
the brain lesions in nmo / nmosd may be located in characteristic regions and present with symptoms mimicking non neurological disease .
we herein present a case of an adult female who was admitted with intractable vomiting and hiccups ; subsequently on mri brain found to have very tiny demyelinating foci in area postrema . |
it has been known that because of application of mechanical force to the tooth crown a tooth can be moved to a desirable spot . the transmission of the force to alveolar bone is mediated by the response of periodontal ligament ( pdl ) , which leads to adaptation of the periodontal tissues to mechanical stress .
the tissue response caused by orthodontic force does occur not only in periodontal tissue but also in dental pulp tissue .
orthodontic force can cause the dental pulp circulatory disturbances and vascular changes [ 2 , 3 ] , which caused the oxygen levels descending in dental pulp ; actually it represented a process of inflammation [ 4 , 5 ] .
as a pathological stimulus , hypoxia will inevitably induce the defensive reaction of dental pulp tissue so as to maintain the stability of internal environment [ 6 , 7 ] .
previous studies indicated that pulp anaerobic can lead to the formation of new blood vessels in the process of orthodontic tooth movement .
angiogenesis , the formation of new blood vessels , is a complex process including extracellular matrix remodeling , secretion of proteolytic enzymes , endothelial cell migration and proliferation , capillary differentiation , and anastomosis .
hypoxia inducible factor-1 ( hif-1 ) , a heterodimeric transcription factor , is composed of ( inducible ) and - ( ubiquitous- ) subunits .
-subunit protein is constitutively present , whereas the stability and transcriptional activity of -subunit are precisely controlled by the intracellular oxygen concentration [ 10 , 11 ] .
recent studies showed that hif-1 mediates the angiogenic response to hypoxia through upregulating the expression of multiple angiogenic factors including vegf and angiopoietins .
vegf is involved in both angiogenesis and osteogenesis which serves various biological functions , such as increasing vascular permeability , promoting monocytic chemotaxis , and regulating endochondral ossification [ 13 , 14 ] .
it was found that when vegf was inactivated in mice , blood vessel invasion was nearly abolished and trabecular bone formation was impaired .
vegf is regarded as the most essential factor for the differentiation of the vascular system among all the proangiogenic factors .
these observations provide direct evidence that vegf plays an important role in bone formation and orthodontic tooth movement . according to previous study
, we found that hif - vegf signaling pathways might be involved in response of dental pulp tissue to mild continuous mechanical force in vitro . in order to analyze the expression and function of hif-1 and vegf in dental pulp in vivo
, we established an animal model of tooth movement to explore how the expression of hif-1 and vegf is affected by stimulating pulp hypoxia in pulp tissue after orthodontic force and to probe into the possible mechanism of pulp tissue to maintain their own stability in the process of orthodontic tooth movement .
the study design was submitted to and approved by the animal ethical committee of shandong university ( number ecaesdusm2012075 ) .
this study contained forty - five male wistar rats , eight weeks of age , with an average weight of 250 20 g , obtained from school of stomatology , shandong university .
animals were fed with a standard diet ( vital river laboratory animal company , beijing , china ) and mineral water ad libitum to avoid any discomfort after the orthodontic appliance inserting .
rats were anesthetized with an intraperitoneal injection of 2.5% tribromoethanol , 0.25 g / kg body weight .
0.032 ) , placed from the right maxillary first molars to the incisors of animals .
we prepared a cervical groove on the incisors , in which the ligature wire was seated and secured with light - cured resin ( z100 , 3 m , sumar , so paulo , brazil ) .
this coil spring was employed for mesial inclination of the first molar . according to previous studies ,
40 or 50 g force was applied in their experimental rat model [ 18 , 19 ] .
however , we thought that a force of 40 or 50 g appears too high , so we applied a force of 30 g in our experiment .
the animals were randomly divided into six groups including one control group not submitted to force application and five experimental groups of 1 , 3 , 7 , 14 , and 28 d of force application .
the tooth movement was measured between the distal surface of the first molar and the mesial surface of the second molar using a vernier caliper with a minimum measurable distance of 0.05 mm .
after the experimental period , the animals were sacrificed by a transcardial perfusion of 4% paraformaldehyde fixing solution .
the maxilla was sectioned in the area of the maxillary first molars and placed in the same fixative for 48 hours .
after completion of decalcification , the blocks of tissue which contain first molars and surrounding alveolar bone were dehydrated in graded alcohol .
then the specimens were embedded in paraffin and serial longitudinal 5 m thick sections were cut in a mesiodistal direction , parallel to the long axis .
the sections were stained with he and examined under light microscopy for analyzing the pulp structures and vascular changes .
sections were deparaffinized in xylene and hydrated in graded alcohol . to block endogenous peroxidase activity ,
then the sections were permeabilized in pbs , using normal goat serum for 20 min to block nonspecific binding . after rinsing
, sections were incubated with monoclonal mouse anti - hif-1 ( 1 : 200 , abcam inc . ,
cambridge , ma ) , monoclonal mouse anti - vegf ( 1 : 200 , abcam inc . ,
next , sections were incubated with biotinylated goat anti - mouse igg ( 1 : 200 , zhongshan golden bridge biological technology co. ltd . ,
the signals were visualized using 3 - 3 diaminobenzidine ( dab , wako , osaka , japan ) and lightly counterstained with hematoxylin . for negative controls ,
all specimens were analyzed under bright - field microscopy ( bx51 , olympus , tokyo , japan ) , and images were captured with a ccd camera ( cs600 , olympus , tokyo , japan ) .
both descriptive and inferential statistical analyses were used to analyze the protein level of hif-1 and vegf .
as shown in table 1 , the distance of tooth movement increased with the extension of experimental period .
the highest mean value of tooth movement was found in the 4 w group .
histological examination showed that tooth movement for different periods presented different changes in pulp tissue structure , as demonstrated by cellular and structural events coupled with inflammatory process .
our study revealed the presence of inflammatory cells throughout the experimental period , indicating an inflammatory process as a response to orthodontic force .
we could observe the inflammatory cells inside the blood vessels and in the pulp connective tissue , such as neutrophils and monocytes , which act as phagocytes and restrict the inflammatory process .
also the edema at pulp tissue was observed in the experimental groups . in the control group ,
the odontoblasts were adjacent to the predentin layer , with the cell bodies around the pulp and the cytoplasm crossing the predentin layer and reaching the dentinal tubules ( figure 1(a ) ) .
compared to control group , the odontoblasts maintained their characteristic organization in all experimental groups , but the nuclei had expanded and diffused chromatin and the cytoplasm was more basophilic , with sparser cells ( figures 1(b)1(f ) ) .
this alteration occurred mainly in the mesial surface of the pulp of the teeth . at the same time , in the control group , we can observe a cell - rich zone interiorly to this cell - free zone which was very clear on the coronal pulp ( figure 1(a ) ) . in the 3 d group ,
the pulp showed normal characteristics , but with changes in the odontoblastic layer in the mesial surface of the coronal pulp ( figure 1(c ) ) .
the central area of the coronal pulp displayed the weil zone in the control group , which is the cell - free zone at the periphery of the dental pulp , while in the experimental groups the weil zone disappeared at the areas in which the odontoblastic layer was more altered obviously and with larger cells .
the pulp tissue swelled and appeared more loose as if some serous fluid accumulated excessively between connective - tissue cells . at 1 d and 3 d of tooth movement ,
the odontoblasts were exhibited normally and the weil zone was once again normally observed on the coronal pulp ( figures 1(b ) and 1(c ) ) . in experimental groups ,
the cell - rich layer was larger than the control group , with characteristic cells such as fibroblasts , undifferentiated mesenchymal cells , and defense cells .
vascularization in the control group was concentrated in the central zone of the coronal and root pulp , characterized by the presence of wider vessels ( figure 1(a ) ) . in the experimental groups ,
vascularization was also concentrated in the central region of the dental pulp and characterized by the presence of wider blood vessels , and it was more closely to the odontoblastic layer .
the 1 d and 3 d groups exhibited areas of congested vessels in the central region of the pulp especially at the root , characterized by the presence of erythrocytes ( figures 1(b ) and 1(c ) ) .
the vessels were still congested at 2 w and often displayed a substance with light purple staining , suggesting the presence of plasma proteins . in the control group and 3 d experimental group
we also observed accessory root canals especially at the furcation region of the molars ( figures 1(a ) and 1(c ) ) .
these root canals contained blood vessels , nerves , and collagen fibers and allowed contact between the pulp tissue and the connective tissue in the periodontal ligament .
the regions of accessory root canals presented a sign of inflammatory reaction at the initial periods of tooth movement .
immunohistochemical analysis showed that the expression of hif-1 and vegf proteins changed obviously under orthodontic force .
hif-1 protein was poorly stained in dental pulp of control group ( figure 2(a ) ) . yet ,
staining of hif-1 was markedly positive in the 1 , 3 , 7 d , and 2 w group ; then it weakened in 4 w group ( figures 2(b)2(f ) ) .
the expression of hif-1 was mainly detected in the nuclei of odontoblasts , pulpal fibroblast , vascular endothelial cells , and mononuclear macrophage under mechanical force .
hif-1 was also observed in the cytoplasm of these cells . in the 1 d and 3 d groups ,
hif-1 expression was slightly stronger compared to the control group . in the 7 d group , expression of hif-1 was stronger than the 3 d group . in the 2 w group ,
hif-1 was strongly expressed in the nuclei and cytoplasm of odontoblasts , pulpal fibroblast , and vascular endothelial cells .
the strongest expression of hif-1 in the nuclei during the whole experiment was detected in this group . in the 4 w group , hif-1 was expressed slightly compared to the 2 w group but was stronger compared to the control group . on the other hand ,
vegf immunostaining was observed in newborn vascular endothelial cells in the control group ( figure 3(a ) ) .
then vegf staining was lightly colored in 1 d group ( figure 3(b ) ) .
subsequently , vegf staining became stronger in the 3 , 7 d , and 2 w groups and it weakened in the 4 w group ( figures 3(c)3(f ) ) .
the expression of vegf was mainly observed in vascular endothelial cells , fibroblasts , and macrophages . in the 3 d group ,
vegf expression was slightly stronger but there was no change when compared to the control group .
following the same kinetics , vegf protein expression increased further up to 2 weeks and its levels reached the peak .
finally , the expression of vegf became weak in 4 w group compared to the 2 w group . in 4 w group , cytoplasmic expression of vegf was slightly expressed but still stronger than the control group .
our present study showed significant vascular changes early in the initial time of tooth movement and an inflammatory process in the dental pulp as a response to orthodontic force .
we observed the initial inflammatory cells inside the blood vessel in the dental pulp tissue . under orthodontic force application , the expression of hif-1 and vegf changed differently to regulate the hypoxic environment in dental pulp .
as we know , the dental pulp is surrounded by a rigid , noncompliant shell and its survival depends on the blood vessels . as a consequence of these unusual environmental constraints , changes in pulpal blood flow or vascular tissue pressure
might bring in serious implications for the health of the dental pulp [ 20 , 21 ] . during the process of tooth movement , periodontal ligament
is compressed in the direction of orthodontic force and the dental pulp was also in a state of stress .
orthodontic forces are known to produce inflammatory reactions as well as cell damage and circulatory disturbances in dental pulp tissue [ 22 , 23 ] ; in fact these are a presentation of local inflammation .
the circulatory disturbances will lead to the oxygen level descending ; thus it creates a hypoxic environment in dental pulp .
then the defensive reaction in dental pulp will happen through stimulating a series of cytokines and signal pathways to regulate the inflammatory reaction and cell damage so that it can maintain the stability of internal environment [ 6 , 7 ] .
in addition , hypoxia is able to induce the inflammation reaction in dental pulp by induction of gene expression in inflammatory cells .
the molecular mechanism of gene expression is related to many mediators and factors ; hif-1 and nuclear factor-b ( nf-b ) are considered as the main of correlative transcription factors .
furthermore , previous study confirmed that hypoxia increased the expression of the proinflammatory cytokines such as interleukin- ( il- ) 1 , il-6 , il-8 , vegf , and tumor necrosis factor- ( tnf- ) .
these proinflammatory cytokines play an important role in orthodontic tooth movement through regulation of matrix metalloproteinase expression and osteoclast differentiation [ 25 , 26 ] . in our experiment , we can observe the initial inflammatory cells inside the blood vessels in the pulp tissue at 1 d and 3 d , such as neutrophils , eosinophils , and monocytes . as we mentioned before , it is a hypoxic environment in the initial phase of orthodontic tooth movement , and inflammatory reaction occurred in the dental pulp tissue due to short - term hypoxia .
many studies have demonstrated that hypoxia has impact on the activity of nf-b , while hypoxia - induced nf-b modulated the levels and activity of hif-1 . on the other hand , some proinflammatory cytokines ( tnf- ,
il-1 , il-6 , and il-8 ) and proinflammatory enzymes ( cyclooxygenase-2 ( cox-2 ) , nitric oxide synthase ) are controlled by nf-b .
also in vitro experiments have showed that hypoxic conditions increased the expression of il-6 , il-8 , cox-2 , and vegf in dental pulp cells .
however , enhanced immune - reactivity of hif-1 is not only caused by hypoxic conditions , but also normoxic conditions , since the promoter of hif-1 is responsive to nf-b and the stabilization of hif-1 is also dependent on nf-b in normoxia [ 25 , 29 , 30 ] . based on the present results in this field , the function and mechanism of hypoxia in dental pulp tissue during the process of orthodontic tooth movement require further investigation . although inflammation is induced by hypoxia , we think that the inflammation may act as a physiological signal for angiogenesis .
vegf , as a proinflammatory cytokine , also has angiogenic activity [ 31 , 32 ] .
angiogenesis or the so - called vascularization is a process of neovascularization , which is a complex process including extracellular matrix remodeling , endothelial cell migration and proliferation , secretion of proteolytic enzymes , capillary differentiation , and anastomosis .
vegf is considered as the most essential factor for the differentiation of the vascular system among all the proangiogenic factors .
as previously stated , hypoxia is a strong inducer of vegf expression [ 33 , 34 ] and vegf is regulated by both osterix and hif-1 .
it is demonstrated that hif-1 is a major regulator of cellular response to hypoxia [ 36 , 37 ] . under hypoxic conditions ,
hif-1 is in a stable state and is translocated to the nucleus where hif-1 binds to its dimerization partner , hif-1 ; then it stimulated the expression of its target genes , such as vegf [ 38 , 39 ] .
additionally , our early study indicated that the expression of vegf increased in dental pulp after application of mechanical force in vitro . in our experiment , we have shown that the expression of hif-1 and vegf changed when they are subjected to hypoxic stress and reached the peak at 2 w under the orthodontic force .
so we speculated that in the early period orthodontic movement induced a hypoxic environment and hypoxia leads to the induction and increasing expression of hif-1 while vegf decreases instead because the blood vessels were closured temporarily . with time increasing , vegf expression increased which is followed by upregulation of hif-1 because the oxygen level continues descending . in turn
, vegf promotes new blood vessels forming and hence brings various necessary nutrients and enhances the level of oxygen to adapt to the low oxygen environment [ 40 , 41 ] .
thus the hypoxic situation was relieved and it increased the oxygen supply of dental pulp tissue so as to keep the balance of internal and external environment and establish the tissue homeostasis . in accordance with our findings , the study published by rmer et al
. also found upregulation of hif-1 expression in the dental pulp tissue of orthodontic moved teeth .
they emphasized the transient immune reactivity of hif-1 during the initial phase of orthodontic treatment in dental pulp .
besides , our results are consistent with previous study , which indicated that hif-1 activated vegf promoter activity and hif-1 upregulates vegf so as to enhance bone remodeling . on the other hand , in our study ,
however , if the force is too small , it is not enough to cause tooth movement and the expression of hif-1 and vegf may be negative .
in contrast , if the force is too high , it would induce the degeneration and necrosis of dental pulp ; thus the expression of hif-1 and vegf would be another case .
therefore , whether the expression of hif-1 and vegf is dependent on force magnitude will need to be further gone into . in summary
, we may conclude that tooth movement caused pulpal tissue alterations coupled with an inflammatory process .
the expression of hif-1 and vegf was enhanced by mechanical force , which indicated that hif-1 and vegf may play an important role in retaining the homeostasis of dental pulp during orthodontic movement .
hif-1 may serve as a global mediator of the angiogenic response to hypoxia by inducing multiple angiogenic factors including vegf . | orthodontic force may lead to cell damage , circulatory disturbances , and vascular changes of the dental pulp , which make a hypoxic environment in pulp . in order to maintain the homeostasis of dental pulp , hypoxia
will inevitably induce the defensive reaction . however , this is a complex process and is regulated by numerous factors . in this study , we established an experimental animal model of orthodontic tooth movement to investigate the effects of mechanical force on the expression of vegf and hif-1 in dental pulp .
histological analysis of dental pulp and expressions of hif-1 and vegf proteins in dental pulp were examined .
the results showed that inflammation and vascular changes happened in dental pulp tissue in different periods .
additionally , there were significant changes in the expression of hif-1 and vegf proteins under orthodontic force .
after application of mechanical load , expression of hif-1 and vegf was markedly positive in 1 , 3 , 7 d , and 2 w groups , and then it weakened in 4 w group .
these findings suggested that the expression of hif-1 and vegf was enhanced by mechanical force .
hif-1 and vegf may play an important role in retaining the homeostasis of dental pulp during orthodontic tooth movement . |
forty middle - aged obese iranian individuals with type ii diabetes ( 19 females and 21 males , aged 44.80 6.04 years , body mass index ( bmi ) 34.15 3.89 kg / m2 ) , who consecutively visited the outpatient clinics for diabetes mellitus in sina university hospital from september 2008 to january 2009 , were enrolled for the present study .
past medical history and clinical data were collected by trained medical worker of the hospital who was supervised by dr .
anthropometric indices including height , weight , hip and waist circumference were taken while subjects were in the standing position and wearing light clothing without shoes .
the waist circumference was taken to the smallest standing horizontal circumference between the lower rib cage and the iliac crest ; the hip circumference was taken as the largest standing horizontal circumference of the buttocks .
these parameters were measured by well - trained dietitians . waist - hip ratio ( whr )
the subjects underwent a detailed examination by the medical office to assess the health status .
the study was reviewed and approved by the institutional ethics committee of tabriz university of medical sciences and written informed consent was obtained from all of the subjects after the procedure had been explained to them .
a venous blood sample ( 5ml ) was drawn from all of the subjects who referred after 12-hour overnight fasting .
type ii diabetes was defined based on history of patients taking oral hypoglycemic drugs or according to the last american diabetes association 's classification in which diabetes is defined as a fasting glucose of more than 126 mg / dl.20 the diabetic patients were treated with oral hypoglycemic agents ( metformin , n=30 ; glibenclamide ; n = ll ) .
the duration of diabetes was 1 to 5years ( mean , 2.80 2.34 years ) .
patients did not consume alcohol or perform heavy exercises for at least 1 week before the study .
the non - diabetic control group consisted of 40 middle - aged non - diabetic obese individuals ( 21 women and 19 men ; aged 43.02 9.00 years ; ( bmi ) 35.25 4.10 kg / m ) who had received an annual health check - up . to select the non - diabetic control individuals ,
the following criteria were used : 1 ) no diabetes in their first degree relatives .
non - diabetic subjects with endocrine disease , significant renal or hepatic diseases , and those receiving medications that control glucose metabolism , hypertension or hyperlipidemia , were excluded from the study .
total cholesterol , triglycerides and high - density lipoprotein - cholesterol ( hdl - c ) were also measured .
low - density lipoprotein - cholesterol ( ldl - c ) was estimated indirectly using the friedewald formula for subjects with a serum tg concentration of less than 400 mg / ml .
fasting serum insulin was measured by enzyme immunoassay using human elisa kit ( q-1-diaplus , usa ) after the serum samples were thawed at room temperature .
this assay had a sensitivity margin of 0.5 iu / ml . intra and inter - assay coefficients of variation were 6.45 and 6.45% , respectively .
hemoglobin aie was measured according to boronate affinity assay by nyco - card hbalc protocol ( norway , with the coefficient of variation ( cv ) below 5% ) from whole blood immediately after obtaining the samples .
according to the percentage of hbalc , diabetic group was sub grouped into the well controlled ( the patients who had hbalc below 8% , n = 25 ) and poorly controlled ( the patients who had hbalc above 8% , n = 15 ) .
serum adiponectin concentration was measured by enzyme - linked - immuno sorbent assay ( elisa ) , using a commercially available kit , human adiponectin elisa kit ( bio vendor laboratory medicine , inc , gmbh ) using specific human adiponectin antibody .
the intra - assay and inter - assay coefficients of variation were 4.4 and 5.7 , respectively . before the assay , quality controls and all sera
body mass index ( bmi ) was calculated as weight ( kilogram ) divided by height squared ( in square meters ) .
body fat content was calculated according to the method of lean et al21 which have been shown to correlate very well with the percentage of body fat measured by underwater weighting13 using the following formula : body fat % ( men ) = [ ( 0.567 waist circumference in cm ) + ( 0.101 age in years ) ] - 31.8 body fat % ( women ) = [ ( 0.438 waist circumference in cm ) + ( 0.221 age in years ) ] - 9.4 serum adiponectin levels have been correlated to body fat percent calculated by this formula.22 insulin resistance and cell function were derived using the homa method23 throuhg the following formulae : insulin resistance ( homa - ir ) = [ fasting insulin ( iu / ml ) fasting glucose ( mmol/1 ) / 22.5 ] cell function ( homa- % ) = [ 20 fasting insulin ( /ml ) / fasting glucose ( mmol/1 ) - 3.5 ] insulin sensitivity was estimated using the quantitative insulin sensitivity check iondex ( quicki ) determined based on the following equation : quicki= 1/ [ log insulin ( iu / ml ) + log glucose ( mg / dl)].24 low quicki indicates low insulin sensitivity , while high quicki indicates high insulin sensitivity .
all continuous data were expressed as mean sd . data analysis were performed using software spss for windows version 14 .
univariate and linear regression analysis were done for determining the relationship between serum adiponectin and other variables and pearson correlation coefficient was obtained .
independent variables including bmi , body fat percent , insulin , hip , fasting glucose , homa - bf , homa - ir and waist were forced into the model .
for all statically assessments , a value of p < 0.05 was statistically accepted as significant .
forty middle - aged obese iranian individuals with type ii diabetes ( 19 females and 21 males , aged 44.80 6.04 years , body mass index ( bmi ) 34.15 3.89 kg / m2 ) , who consecutively visited the outpatient clinics for diabetes mellitus in sina university hospital from september 2008 to january 2009 , were enrolled for the present study .
past medical history and clinical data were collected by trained medical worker of the hospital who was supervised by dr .
anthropometric indices including height , weight , hip and waist circumference were taken while subjects were in the standing position and wearing light clothing without shoes .
the waist circumference was taken to the smallest standing horizontal circumference between the lower rib cage and the iliac crest ; the hip circumference was taken as the largest standing horizontal circumference of the buttocks .
these parameters were measured by well - trained dietitians . waist - hip ratio ( whr )
the subjects underwent a detailed examination by the medical office to assess the health status .
the study was reviewed and approved by the institutional ethics committee of tabriz university of medical sciences and written informed consent was obtained from all of the subjects after the procedure had been explained to them .
a venous blood sample ( 5ml ) was drawn from all of the subjects who referred after 12-hour overnight fasting .
type ii diabetes was defined based on history of patients taking oral hypoglycemic drugs or according to the last american diabetes association 's classification in which diabetes is defined as a fasting glucose of more than 126 mg / dl.20 the diabetic patients were treated with oral hypoglycemic agents ( metformin , n=30 ; glibenclamide ; n = ll ) .
the duration of diabetes was 1 to 5years ( mean , 2.80 2.34 years ) .
patients did not consume alcohol or perform heavy exercises for at least 1 week before the study .
the non - diabetic control group consisted of 40 middle - aged non - diabetic obese individuals ( 21 women and 19 men ; aged 43.02 9.00 years ; ( bmi ) 35.25 4.10 kg / m ) who had received an annual health check - up . to select the non - diabetic control individuals ,
the following criteria were used : 1 ) no diabetes in their first degree relatives .
non - diabetic subjects with endocrine disease , significant renal or hepatic diseases , and those receiving medications that control glucose metabolism , hypertension or hyperlipidemia , were excluded from the study .
total cholesterol , triglycerides and high - density lipoprotein - cholesterol ( hdl - c ) were also measured .
low - density lipoprotein - cholesterol ( ldl - c ) was estimated indirectly using the friedewald formula for subjects with a serum tg concentration of less than 400 mg / ml .
fasting serum insulin was measured by enzyme immunoassay using human elisa kit ( q-1-diaplus , usa ) after the serum samples were thawed at room temperature .
this assay had a sensitivity margin of 0.5 iu / ml . intra and inter - assay coefficients of variation were 6.45 and 6.45% , respectively .
hemoglobin aie was measured according to boronate affinity assay by nyco - card hbalc protocol ( norway , with the coefficient of variation ( cv ) below 5% ) from whole blood immediately after obtaining the samples .
according to the percentage of hbalc , diabetic group was sub grouped into the well controlled ( the patients who had hbalc below 8% , n = 25 ) and poorly controlled ( the patients who had hbalc above 8% , n = 15 ) .
serum adiponectin concentration was measured by enzyme - linked - immuno sorbent assay ( elisa ) , using a commercially available kit , human adiponectin elisa kit ( bio vendor laboratory medicine , inc , gmbh ) using specific human adiponectin antibody .
the intra - assay and inter - assay coefficients of variation were 4.4 and 5.7 , respectively . before the assay , quality controls and all sera
body mass index ( bmi ) was calculated as weight ( kilogram ) divided by height squared ( in square meters ) .
body fat content was calculated according to the method of lean et al21 which have been shown to correlate very well with the percentage of body fat measured by underwater weighting13 using the following formula : body fat % ( men ) = [ ( 0.567 waist circumference in cm ) + ( 0.101 age in years ) ] - 31.8 body fat % ( women ) = [ ( 0.438 waist circumference in cm ) + ( 0.221 age in years ) ] - 9.4 serum adiponectin levels have been correlated to body fat percent calculated by this formula.22 insulin resistance and cell function were derived using the homa method23 throuhg the following formulae : insulin resistance ( homa - ir ) = [ fasting insulin ( iu / ml ) fasting glucose ( mmol/1 ) / 22.5 ] cell function ( homa- % ) = [ 20 fasting insulin ( /ml ) / fasting glucose ( mmol/1 ) - 3.5 ] insulin sensitivity was estimated using the quantitative insulin sensitivity check iondex ( quicki ) determined based on the following equation : quicki= 1/ [ log insulin ( iu / ml ) + log glucose ( mg / dl)].24 low quicki indicates low insulin sensitivity , while high quicki indicates high insulin sensitivity .
all continuous data were expressed as mean sd . data analysis were performed using software spss for windows version 14 .
univariate and linear regression analysis were done for determining the relationship between serum adiponectin and other variables and pearson correlation coefficient was obtained .
independent variables including bmi , body fat percent , insulin , hip , fasting glucose , homa - bf , homa - ir and waist were forced into the model .
for all statically assessments , a value of p < 0.05 was statistically accepted as significant .
forty middle - aged obese iranian individuals with type ii diabetes ( 19 females and 21 males , aged 44.80 6.04 years , body mass index ( bmi ) 34.15 3.89 kg / m2 ) , who consecutively visited the outpatient clinics for diabetes mellitus in sina university hospital from september 2008 to january 2009 , were enrolled for the present study .
past medical history and clinical data were collected by trained medical worker of the hospital who was supervised by dr .
anthropometric indices including height , weight , hip and waist circumference were taken while subjects were in the standing position and wearing light clothing without shoes .
the waist circumference was taken to the smallest standing horizontal circumference between the lower rib cage and the iliac crest ; the hip circumference was taken as the largest standing horizontal circumference of the buttocks .
these parameters were measured by well - trained dietitians . waist - hip ratio ( whr )
the subjects underwent a detailed examination by the medical office to assess the health status .
the study was reviewed and approved by the institutional ethics committee of tabriz university of medical sciences and written informed consent was obtained from all of the subjects after the procedure had been explained to them .
a venous blood sample ( 5ml ) was drawn from all of the subjects who referred after 12-hour overnight fasting .
type ii diabetes was defined based on history of patients taking oral hypoglycemic drugs or according to the last american diabetes association 's classification in which diabetes is defined as a fasting glucose of more than 126 mg / dl.20 the diabetic patients were treated with oral hypoglycemic agents ( metformin , n=30 ; glibenclamide ; n = ll ) .
the duration of diabetes was 1 to 5years ( mean , 2.80 2.34 years ) .
patients did not consume alcohol or perform heavy exercises for at least 1 week before the study .
the non - diabetic control group consisted of 40 middle - aged non - diabetic obese individuals ( 21 women and 19 men ; aged 43.02 9.00 years ; ( bmi ) 35.25 4.10
kg / m ) who had received an annual health check - up . to select the non - diabetic control individuals ,
the following criteria were used : 1 ) no diabetes in their first degree relatives .
non - diabetic subjects with endocrine disease , significant renal or hepatic diseases , and those receiving medications that control glucose metabolism , hypertension or hyperlipidemia , were excluded from the study .
triglycerides and high - density lipoprotein - cholesterol ( hdl - c ) were also measured .
low - density lipoprotein - cholesterol ( ldl - c ) was estimated indirectly using the friedewald formula for subjects with a serum tg concentration of less than 400 mg / ml .
fasting serum insulin was measured by enzyme immunoassay using human elisa kit ( q-1-diaplus , usa ) after the serum samples were thawed at room temperature .
this assay had a sensitivity margin of 0.5 iu / ml . intra and inter - assay coefficients of variation were 6.45 and 6.45% , respectively .
hemoglobin aie was measured according to boronate affinity assay by nyco - card hbalc protocol ( norway , with the coefficient of variation ( cv ) below 5% ) from whole blood immediately after obtaining the samples . according to the percentage of hbalc , diabetic group was sub grouped into the well controlled ( the patients who had hbalc below 8% , n = 25 ) and poorly controlled ( the patients who had hbalc above 8% , n = 15 ) .
serum adiponectin concentration was measured by enzyme - linked - immuno sorbent assay ( elisa ) , using a commercially available kit , human adiponectin elisa kit ( bio vendor laboratory medicine , inc , gmbh ) using specific human adiponectin antibody .
the intra - assay and inter - assay coefficients of variation were 4.4 and 5.7 , respectively . before the assay , quality controls and all sera
body mass index ( bmi ) was calculated as weight ( kilogram ) divided by height squared ( in square meters ) .
body fat content was calculated according to the method of lean et al21 which have been shown to correlate very well with the percentage of body fat measured by underwater weighting13 using the following formula : body fat % ( men ) = [ ( 0.567 waist circumference in cm ) + ( 0.101 age in years ) ] - 31.8 body fat % ( women ) = [ ( 0.438 waist circumference in cm ) + ( 0.221 age in years ) ] - 9.4 serum adiponectin levels have been correlated to body fat percent calculated by this formula.22 insulin resistance and cell function were derived using the homa method23 throuhg the following formulae : insulin resistance ( homa - ir ) = [ fasting insulin ( iu / ml ) fasting glucose ( mmol/1 ) / 22.5 ] cell function ( homa- % ) = [ 20 fasting insulin ( /ml ) / fasting glucose ( mmol/1 ) - 3.5 ] insulin sensitivity was estimated using the quantitative insulin sensitivity check iondex ( quicki ) determined based on the following equation : quicki= 1/ [ log insulin ( iu / ml ) + log glucose ( mg / dl)].24 low quicki indicates low insulin sensitivity , while high quicki indicates high insulin sensitivity .
univariate and linear regression analysis were done for determining the relationship between serum adiponectin and other variables and pearson correlation coefficient was obtained .
independent variables including bmi , body fat percent , insulin , hip , fasting glucose , homa - bf , homa - ir and waist were forced into the model .
for all statically assessments , a value of p < 0.05 was statistically accepted as significant .
table 1 summarized the anthropometric , metabolic and clinical characteristics of non - diabetics and type ii diabetes mellitus subjects .
the mean age of diabetic group was 44.80 6.04 years and non - diabetic group was 43.02 9.00 years .
the mean of bmi for diabetic and no - diabetic groups was 34.15 3.89 kg / m and 35.25 4.10 kg / m , respectively ( p > 0.05 ) .
when we compared diabetic and no - diabetic groups regarding age , bmi , height , weight , whr , waist , total cholesterol and insulin , no difference was observed ( table 1 ) .
however , the mean concentration of hdl - c , adiponectin , homa- percent and quicki index in control group were significantly higher than diabetic group ( p < 0.05 ) .
on the other hand , the mean concentrations of fasting blood glucose , triglyceride , ldl - c , hbalc , systolic and diastolic blood pressure and homa - ir index in diabetic group was significantly higher than control group ( p < 0.05)(table 1 ) . anthropometric and metabolic
parameters of diabetic and non - diabetic groups in all of the study subjects , mean basal adiponectin concentration was 5.49 2.38 ( range 2.10 - 14.01 g / ml ) .
mean ( sd ) serum adiponectin concentration was significantly lower in subjects with t2 dm compare to non - diabetic control group ( 4.55 1.88 vs. 6.44 2.47 ( g / ml ) ( p < 0.0001 ) .
this was observed both in males ( p = 0.006 ) and females ( p = 0.013 ) of subjects with type ii diabetes compare to males and females in the control group .
serum adiponectin levels of non - diabetic control subjects ranged from 3.18 to 14.01 g / ml .
men had a mean serum adiponectin level of 4.76 1.91 g / ml ( table 1 ) . despite no difference in mean bmi ( all the females , 35.29 4.23 kg / m ; all the males , 34.11 3.74
kg / m ) mean plasma concentrations of women were almost 1.5 compared with men ( all the females , 6.23 2.59 ( g / ml ; all the males , 4.76 1.91 g / ml ) .
moreover , in sex adjusted analysis , the mean concentration of serum adiponectin in diabetic group ( 5.18 2.08 vs. 3.99 1.570 g / ml ) and non - diabetic group ( 7.18 2.68 vs. 5.61 0.57 g / ml ) females was significantly higher than males in the both groups ( p < 0.05 ) .
this increase in women was also seen when the levels were normalized based on bmi when expressed in terms of adiponectin / bf percent , the increase in women was less pronounced ( 1.07-times that of men compared to 1.28-times seen in adiponectin / bmi ) .
we have calculated body fat content ( % ) based on the method of lean et al using the formula involving age and waist circumference .
the body fat percent of non - diabetic and diabetic subjects was 42.05 7.94% and 39.76 7.49% , respectively .
body fat percent of women was about 1.5-times higher than that of men in diabetic and non - diabetic groups .
as expected , homa calculations showed that type ii diabetic subjects had a significant increase in insulin resistance ( p = 0.001 , age and bmi adjusted data ) and impaired cell function ( p < 0.001 ) .
furthermore , homa- cell function was lower in the poorly - controlled diabetic ( 68.54 41.46 ) than well - controlled ( 125.61 74.50 ) subgroup ( p = 0.05 ) .
it was suggested that this subgroup had a more severe deterioration of their cell function than the other subgroup .
homa- cell function was also lower in male compared to female in diabetic group ( 85.03 42.65 vs. 126.19 87.23 ) .
( a ) represents the relationship between serum adiponectin levels and hdl - c in all the study subjects .
adiponectin was positively correlated with hdl - c ( r = 0.385 , p = 0.001 ) in all the subjects including males ( r = 0.379 ; p = 0.016 ) and females ( r = 0.412 ; p = 0.002 ) . on the other hand ,
adiponectin was negatively correlated with waist - hip ratio(r = -0.338 ; p = 0.002 ) only in the total subjects as represented in figure 1 ( b ) .
adiponectin showed a correlation with waist circumference only in females ( r = -0.455 ; p = 0.002 ) as seen in figure 1 ( c ) .
fasting serum adiponectin levels of normal women ( filled triangles ) upper dashed line andnormal men ( empty triangles ) , lower dashed line and total subjects middle solid line , were plotted against hdl - c .
b. relationship between serum adiponectin levels and waist to hip ratio in all subjects . c. relationship between serum adiponectin levels and waist circumference in women .
serum adiponectin levels of women were reduced approximately as 50% in poorly - controlled diabetics ( from 7.18 2.68 to 3.95 .
serum adiponectin levels were lower as 50% ( from 5.61 1.96 to 2.90 0.63 g / ml p = 0.001 ) in poorly - controlled diabetics men compared to non - diabetic control males .
there was no significant change in serum adiponectin in well - controlled diabetic women and men compared to non - diabetic control subjects .
serum adiponectin exhibited approximately 40% reduction in poorly - controlled diabetic women ( from 0.21 0.09 to 0.12 0.04 g / unit bmi ; p < 0.001 ) compared to normal women ; while there was no significant change in well - controlled diabetic men .
in the males , serum adiponectin levels were lower as 50% in poorly - controlled group ( from 0.16 0.06 to 0.09 0.03 g / unit bmi ) .
adiponectin levels were represented for men ( empty bar ) and for women ( filled bar ) .
adiponectin levels were significantly reduced in well ( p=0.02 ) and poorly controlled diabetic compared to control group , ( p<00001 ) . among women , adiponectin levels
were significantly reduced in poorly controlled diabetic compared to control women ( p=0.010 ) . among men adiponectin levels normalized to bmi
were significantly reduced in poorly controlled diabetic compared to control men ( p=0.001 ) . in all the subjects-level ( p = 0.001 ) in univariate analysis using adiponectin as the dependent variable in total study subjects , hbalc , gender , glucose , waist size , waist - hip ratio , dbp , hbalc , tg , hdl - c and quicki predicted the variability in adiponectin significantly ( p < 0 .
in total number of the subjects ( 80 ) , simple linear regression analysis yielded highest correlation of serum adiponectin to hdl - c ( r = 0.375 ; p = 0.001 ) , waist circumference ( r = -0.345 ; r = 0 .
001 ) and lastly to hbalc ( r = -0.245 ; p = 0.028 ) . in the diabetic subjects gender ( r = -0.399 ;
p = 0.011 ) and waist ( r = -0.317 ; p = 0.046 ) was related to adiponectin . on the other hand , in non - diabetic subjects , hdl - c ( r= 0.337 ; p=0.034 ) and quicki ( r = 0.312 ; p = 0.05 ) were related to adiponectin in addition to waist ( r = -0.451 ; p=0.003 ) and gender , ( r = -0.320 ; p = 0.044 ) . therefore , the main differences due to diabetic status were not a significant relationship between adiponectin and good profile lipid ( hdl - c and ouicki ) .
thus , serum adiponectin levels negatively dependent upon gender and waist size as a central obesity factor in the two groups .
these results suggested that adiponectin levels of non - diabetics were influenced differently from the diabetic groups . in non - diabetic group , adiponectin levels
were partly contributed by increased hdl - c and quicki as an insulin sensitivity marker .
table 2 represents the results of linear regression analysis carried out using serum adiponectin as the dependent variable . in this analysis ,
only those variables were considered which had a significant correlation in the linear regression analysis . in all the total subjects ,
serum adiponectin levels were contributed by hdl - c , waist size and hbalc . in addition , in non - diabetic subjects , hdl - c and waist were the predictor of adiponectin levels .
on the other hand , in diabetic subjects , serum adiponectin levels were also predicted by waist size .
gender - specific regression analysis revealed that in women , changes in adiponectin levels were explained by waist ( r = -0.455 ; p=0.002 ) and hdl - c ( r = 0.412 ; p = 0.002 ) while in men , adiponectin levels were explained only by hdl - c ( r = 0.379 ; p = 0.016 ) .
these results suggested that men 's adiponectin levels were influenced differently from women . in women , adiponectin levels
were partly contributed by increased waist circumference , an indicator of abdominal obesity , which in turn influenced adiponectin levels .
while other studies have reported differences in leptin and adiponectin levels between diabetic and non - diabetic subjects2526 we decided to investigate the difference in adiponectin levels in obese diabetic and non - diabetic subjects matched for age , gender , and bml there was a significant difference in serum adiponectin concentrations according to gender and presence of diabetes .
gender differences in leptin levels were well recognized;26 however , a few studies ( not all of them ) have re - ported gender differences in adiponectin levels.2728 this study revealed two important findings .
first ; it confirmed previous findings that obesity and type ii diabetes were associated with low serum adiponectin concentrations .
second , our results showed that the serum adiponectin concentration was more closely related to obesity than to insulin sensitivity and fasting insulinemia , which suggested that hypoadiponectinemia in subjects with obesity and type ii diabetes was in at least attributable to obesity indices .
furthermore , our data showed that , in addition to overall obesity , central obesity was an independent negative predictor of serum adiponectin and suggested that adiponectin may represent a link between central obesity and type ii diabetes adiposity ( bmi , lipid mass , percentage of fat ) has been previously reported to be inversely associated with serum adiponectin in japanese,2830 pima indians2531 and caucasians.1425 the etiology of the decreased adiponectin mrna expression in adipose tissue2632 and the lower serum adiponectin levels had been reported in obese subjects25 is not completely understood .
however , adipose tissue - derived cytokines have been suggested that negatively regulate adiponectin synthesis and release . in particular , tnf- and interleukin-6 have been shown to reduce adiponectin mrna content and inhibit adiponectin release in 3t3-l1 cells.3334 another possibility is a reduction in metabolic function of the adipocyte as it ages or hypertrophies .
for example , aged 3t3l1 cells contain more triglycerides and are not only more insulin resistant , but also have decreased adiponectin gene expression compared to young adipocytes.35 in addition to confirming the association between some of the obesity indices and adiponectin , we found that central obesity was an independent negative predictor of serum adiponectin level .
the relationship of adiponectin with whr , and waist appeared to be stronger than other obesity indices or bmi , indicating that central fat distribution is a better determinant of circulating adiponectin than total fat mass .
similar to our findings , cnop et al14 have reported recently that intra - abdominal fat ( but not bmi ) was significantly and independently associated with adiponectin , whereas a japanese study36 has reported an inverse association between adiponectin and whr in morbidly obese patients but not in overweight and moderately obese patients .
we confirmed that adiponectin levels are related to triglyceride levels and there was a positive and strong correlation between serum adiponectin and hdl - cholesterol in the two groups , as previously reported in japanese subjects.1828 matsbara et al29 have reported a strong positive correlation between adiponectin and hdl - c independently from visceral at , explaining 37% of the variance in hdl cholesterol .
our results are also in accordance with the previous study that has shown a strong correlation between plasma adiponectin levels and hdl cholesterol independently of age , gender , bmi , and fasting insulin concentration.37 in that study , adiponectin appeared to predict hdl cholesterol level in patients with type ii diabetes , independent from common metabolic risk factors.37 thus , it is possible that adiponectin , like hdl cholesterol , could eventually be considered as an independent protective cardiovascular risk factor.37 such a strong correlation between adiponectin and hdl cholesterol levels may likely be explained by the activation of peroxisome proliferator - activated receptor- which influences the expression of genes encoding for proteins involved in hdl metabolism.36 for instance , adiponectin has been reported to increase peroxisome proliferator - activated receptor- ligand activities both in skeletal muscle and liver33 leading to an increased synthesis of hdl cholesterol .
our observations , regarding the impact of sex , suggested that adiponectin concentration was also related to factors independent of obesity indices . at any obesity indices , adiponectin concentrations
furthermore , in men and women pair - matched for age , and bmi adiponectin concentrations were greater in women in non - diabetic and diabetic groups .
earlier reports have revealed the sexual dimorphism of adiponectin , with females having significantly higher circulating levels of total adiponectin than males in both humans and rodents.27 xu et al,38 showed that the sexual dimorphism of adiponectin is mainly due to the difference in its hmw ( high molecular weight ) form in both human and rodents , whereas females and males have comparable levels of the mmw ( medium molecular weight ) and lmw ( low molecular weight ) forms . in line with this result , the recent study using velocity ultra - centrifugation also observed that the percentage composition of hmw adiponectin in female mice was significantly higher than male mice.39 clinical studies have found that decreased ratios of hmw / total adiponectin were closely associated with the development of diabetes and cardiovascular diseases.4041 given the important role of hmw adiponectin in increasing insulin sensitivity and in preventing atherosclerosis , the sexual difference in hmw adiponectin might partly explain the fact that men have a higher incidence of atherosclerosis and are more susceptible to insulin resistance than women.38 in addition , in line with another study42 in our study , neither homa - ir and homa- cell function nor quicki were significantly different between males and females in the two groups . in brief , we speculated that low serum adiponectin levels in obese type ii diabetes and even lower levels in subjects who had poorly controlled diabetes was likely due to central obesity indices rather than insulin resistance and hyperinsulinemia .
gm coordinated the study , carried out the design , analyzed the data and prepared the manuscript .
nz provided assistance in the design of the study , coordinated all the experiments and participated in manuscript preparation . | background : adiponectin is an adipocyte secreted protein with important biological functions hypoadiponectinemia is associated with obesity , insulin resistance , and type ii diabetes this study aimed to evaluate serum adiponectin level in obese subjects with type ii diabetes and its correlation with metabolic parametersmethods : this cross - sectional study was performed on 40 obese subjects with type ii diabetes and 40 non - diabetic obese control subjects fasting lipid profile was measured by the enzymatic methods the nycocard hba1c protocol was used to measure hbalc the serum adiponectin , insulin and glucose levels were measured using an enzyme immunoassay and glucose oxidase methods respectivelyresults : type ii diabetes was associated with hypoadiponectinemia , in both men and women serum adiponectin level in non - diabetic subjects ( 6.44 2.47 g / ml ) was significantly higher than diabetics ( 4.55 1.88 g / ml ) furthermore , serum adiponectin concentration in females was significantly higher than males in non - diabetics ( 7.18 2.68 vs 5.61 0.57 ) and diabetic groups ( 5.18 2.08 vs 3.99 1.5 ) there was a negative and significant correlation between serum adiponectin level with waist ( r = -0.451 , p = 0.003 ) , waist to hip ratio ( r = -0.404 , p = 0.01 ) and bmi ( r = - 0.322 , p = 0.042 ) and a positive correlation with hdl ( r = 0.337 , p = 0.034 ) in non - diabetic group in diabetic group , there was only found a negative correlation between adiponectin and waist size ( r = -0.317 , p = 0.046)conclusions : obesity and type ii diabetes are associated with low serum adiponectin concentration |
over recent decades , resistance to antimicrobials has become a worldwide public health problem , and particularly important regarding bacterial agents that cause healthcare related infections.1 today , there are bacteria responsible for infections in hospital environments that are resistant to all existing classes of antimicrobials , which shows their great potential for evolutionary adaptation.2 this , together with the declining numbers of new antimicrobials that are being certified and introduced into clinical practice , is making the growing prevalence of these bacteria with high levels of resistance a subject of increasing concern.3 the formerly abundant flow of new antibiotics of ever - broader spectrum supplied by the pharmaceutical industry has been shown to be a non - renewable resource.4 the options for attempting to control the dissemination of bacterial resistance within hospital settings include educational policies aimed at better adherence to infection control practices among professionals , particularly with regard to hand hygiene ; and strategies for rationalizing the prescription of antimicrobials within hospitals.57 it has now become clear that the use of antimicrobials favors the emergence of resistant bacterial strains .
however , it is also known that the genetic mechanism used by bacteria to acquire resistance to antibiotics not only promotes their spread within hospital environments but also confers stability on the resistance genes , even subsequently , in situations of absence of exposure to antimicrobials.8 this interaction between antibiotic consumption and the development of bacterial resistance to them is of particular interest with regard to gram - negative bacilli ( gnb ) .
the resistance development pattern of gnb today provides a great challenge in treating infections caused by these bacteria .
given that new therapeutic options for treating infections caused by these gnb with high levels of resistance are not expected to become available in the near future , it becomes imperative to study measures that might be capable of improving the antimicrobial susceptibility of these bacterial agents , thereby making it possible to use the drugs that are available .
studies showing the effects of modifications to antibiotic prescription patterns are especially of interest in relation to combating the emergence of resistance in gnb strains.6 the objective of this study was to assess the impact of restriction of cefepime use on antimicrobial susceptibility among the gram - negative bacilli ( gnb ) most frequently involved in healthcare - associated infections at a tertiary - level orthopedic hospital .
this observational study of an ecological nature was conducted at the institute of orthopedics and traumatology , hospital das clnicas , university of so paulo school of medicine , which provides specialized high - complexity tertiary - level care within the field of orthopedics and traumatology in the city of so paulo , brazil .
it was approved by the local ethics committee under the number 0649/07 . at the time covered by this study ,
the hospital had an average of 150 beds available for operations , of which ten were for intensive care . in may 2007 , the standard empirical antibiotic therapy for surgical site infections in this hospital was changed .
this is the main type of hospital infection seen at this hospital , because of the attendance profile . up to this date ,
combination therapy with vancomycin and cefepime was the first choice for empirical treatment of these infections until results had been obtained from cultures , and the consumption of both of these drugs was very high .
after this date , a regime using teicoplanin and amikacin became the standard for empirical antibiotic therapy in such cases , and the use of cefepime was restricted . for the present study , data relating to hospital occupancy and mortality profiles ; incidence of healthcare - associated infection cases ; incidence of gnb as the cause of healthcare - associated infection ; consumption of antimicrobials in the hospital ( in defined daily doses [ ddd]/1000 patient - days ) ; and antimicrobial susceptibility for gnb relating to healthcare infection were obtained and compared between two periods : a 24-month period preceding the restriction on cefepime use , from may 2005 to may 2007 ( first period ) ; and a 24-month period subsequent to introducing the restriction , from may 2007 to may 2009 ( second period ) . for this study , the criteria for defining nosocomial infection recommended by the centers for disease control and prevention ( cdc ) in 2004 were used.10 for the analysis , only the gnb for which more than 30 isolates were obtained in each of the two study periods were selected , in accordance with the norms recommended by the clinical and laboratory standards institute ( clsi ) for analysis and interpretation of cumulative antibiograms.11 the institutional policies for controlling healthcare - associated infections remained the same throughout the two periods . to meet the study objectives ,
the consumption of each antimicrobial was described separately and per class using summary measurements for each of the two periods ( prior and subsequent to the restriction of cefepime use ) , and comparisons were made between the periods using the nonparametric mann
whitney test.12 hospital occupancy and mortality measurements were described and compared in the same manner .
measurements of the antimicrobial susceptibility for each of the selected microorganisms were made according to the period , using absolute and relative frequencies , and the presence of associations in each period was investigated by means of the chi - square test , or by means of the fisher exact test when the sample was very small.12 the tests were conducted using a significance level of 5% .
there were no differences between the two periods regarding the mean length of hospital stay or the mean hospital mortality rate , and there was no significant difference in the incidence of healthcare - associated infection cases between the two periods .
the incidence of gnb as the cause of healthcare - associated infection also did not vary significantly between the two study periods ( table 1 ) . with regard to antimicrobial consumption
, there was a notable and significant drop in the consumption of cefepime after its use was restricted , and a significant increase in the consumption of amikacin .
other antibiotics that presented statistically significant increased consumption after cefepime use was restricted were aztreonam , ertapenem and levofloxacin .
the consumption of imipenem and colistin was lower , in relation to the first study period , after cefepime use was restricted ( table 2 ) .
the gnb with occurrences of more than 30 isolates ( and for which antimicrobial susceptibilities were therefore analyzed ) were the same in the two study periods : acinetobacter baumanii , pseudomonas aeruginosa , klebsiella pneumoniae and enterobacter spp . in the second period ( after cefepime use
was restricted ) , it was observed that the susceptibility for a. baumanii improved in relation to gentamicin and worsened in relation to imipenem . for p. aeruginosa
, there was no change in antimicrobial susceptibilities after cefepime use was restricted . for k. pneumoniae and enterobacter spp , there were improvements in the susceptibility relating to ciprofloxacin , after cefepime use was restricted .
table 3 shows comparisons of the antimicrobial susceptibilities for the gnb studied in the two study periods .
the growing resistance to antimicrobials presented by the bacterial agents involved in healthcare - associated infections has been shown to be a very important problem over recent decades , particularly with regard to gnb.1,3 strategies for rationalizing antimicrobial prescriptions within hospital environments and for changing the pattern of prescriptions for this class of drugs have been used as tools for combating the constant emergence of resistance among gnb.6 analysis of the antimicrobial consumption data in the two study periods showed firstly that the policy of restriction of cefepime use that was instituted in the hospital was effective , considering that the mean monthly consumption of this antibiotic went down from 159.57 ddd per 1000 patient - days in the first period to 13.45 ddd per 1000 patient - days in the second period , and this change was statistically significant . the observed significant increase in amikacin consumption in the second period , from a monthly mean of 13.03 ddd per 1000 patient - days to 93.79 ddd per 1000 patient - days
can be explained by its role in the second study period as a standard antibiotic , replacing cefepime , for empirical treatment of surgical site infections .
this , in turn , correlated more with the increased consumption of ertapenem in the second study period than with the restriction on cefepime use . in the hospital where this study was conducted ,
ertapenem was introduced in march 2006 as the antibiotic of choice for treating infections caused by extended spectrum beta lactamase ( esbl)-producing enterobacteriaceae , and the results from the impact of this introduction , particularly regarding the antimicrobial susceptibility of p. aeruginosa , have already been published.1315 analysis of the consumption of aztreonam , colistin and levofloxacin showed that although there was a statistically significant change between the two periods , the consumption of these drugs was very low throughout the study , compared with the consumption of other antibiotics , thus diminishing the importance of this finding .
on the other hand , the consumption of ciprofloxacin went up from a monthly mean of 38.61 ddd per 1000 patient - days to 50.89 ddd per 1000 patient - days after restriction of cefepime use .
this increasing trend may be related to changes in the sensitivity of gnb in the second study period , since there were improvements in the antimicrobial susceptibilities of k. pneumoniae and enterobacter spp in the period following the restriction of cefepime use . for a. baumanii
, the analysis of the antimicrobial susceptibility showed that there was a significant improvement in susceptibility in relation to gentamicin and worsening in relation to imipenem , although there were no differences in the consumption of these antimicrobials in the hospital . despite the restriction on cefepime use , there was no change in the susceptibility of a. baumanii in relation to this antimicrobial .
regarding the susceptibility of p. aeruginosa , analysis and comparison of the data from the two study periods showed that there was no change after cefepime use was restricted , for any of the antibiotics tested .
for k. pneumoniae and enterobacter spp , comparison of the susceptibilities data showed that there were significant improvements in the susceptibilities of these bacterial agents in relation to ciprofloxacin , in the second study period , although the consumption of this antimicrobial increased in this period .
comparison between these results and those from previous studies , regarding the impact of changes in antimicrobial prescriptions on the sensitivity profile of gnb involved in hospital infections shows the complexity of this interaction , given that changes in the use of a single class of antimicrobials may produce different changes in relation to the susceptibilities of the bacterial agents studied.6,1619 between the two study periods , there was no difference in the patients mean length of hospital stay or in the hospital mortality rate .
these data make it possible to infer indirectly that the severity of the hospitalized patients conditions was probably similar between the two periods , and that the prevalence of nosocomial infection among the hospitalized patients was also similar between the two periods .
this is important because differences in the severity of patients conditions or in the prevalence of nosocomial infection among patients may influence the variations in bacterial susceptibilities described above
. the limitations of ecological studies for analyzing relationships between antimicrobial consumption and the development of bacterial resistance within hospital environments have already been explored.20 bacterial resistance is directed by the principles of natural selection , and its underlying selection and dispersion mechanisms are matters of great complexity .
the influence of antimicrobial consumption on this resistance may even have different effects over the course of time , and will be most important at the time when a specific resistance mechanism appears . regarding the analysis of bacterial susceptibilities , the cutoff points established by the clsi for defining resistance , as used in these types of study , are very high and exclude bacterial strains with low resistance levels from the analysis .
such strains usually precede strains with high resistance levels and would also be important in analyses of hospital environments.21 one limitation in particular in this study was the absence of molecular biology analysis of the bacteria involved , which would have made it possible to evaluate whether the observed differences in antimicrobial susceptibility were caused by the introduction of new bacterial strains or by clonal expansion .
another deficiency was the lack of measurements on healthcare professionals adherence to hospital infection control practices in the two study periods .
although the recommendations for such practices remained the same throughout the two periods , differences in adherence to the recommendations may have influenced the results obtained .
the results from this study suggest that the restriction on cefepime use had a positive impact on the sensitivity profiles of the k. pneumoniae and enterobacter spp strains involved in healthcare - associated infections at the orthopedic hospital where this study was conducted , given that after the restriction was introduced , the susceptibility to ciprofloxacin improved .
however , for a. baumanii , the results suggest that there was a negative impact , given the worsening of susceptibility to imipenem . | introduction : in recent decades , antimicrobial resistance has become a public health problem , particularly in cases of healthcare - associated infections .
interaction between antibiotic consumption and resistance development is of particular interest regarding gram - negative bacilli , whose growing resistance has represented a great challenge.objective:assess the impact of restriction of cefepime use on antimicrobial susceptibility among the gram - negative bacilli ( gnb ) most frequently involved in healthcare - associated infections ( hai).methods : data relating to hospital occupancy and mortality rates , incidence of hai , incidence of gnb as causative agents of hai , antimicrobial consumption at the hospital and antimicrobial susceptibility of gnb related to hai were compared between two periods : a 24-month period preceding restriction of cefepime use and a 24-month period subsequent to this restriction.results:there was a significant drop in cefepime consumption after its restriction .
susceptibility of acinetobacter baumanii improved relating to gentamicin , but it worsened in relation to imipenem , subsequent to this restriction . for pseudomonas aeruginosa , there was no change in antimicrobial susceptibility . for klebsiella pneumoniae and enterobacter spp , there were improvements in susceptibility relating to ciprofloxacin.conclusion:restriction of cefepime use had a positive impact on k. pneumoniae and enterobacter spp , given that after this restriction , their susceptibilities to ciprofloxacin improved .
however , for a. baumanii , the impact was negative , given the worsening of susceptibility to imipenem . |
endoscopy is the most accurate and beneficial way for the diagnosis of dyspepsia , peptic ulcers , and malignancies .
sedative drugs are used for both patient and physician satisfaction during the procedure ; type of endoscopy , duration , degree of difficulty , and patients physical status are the criteria which determine the suitable type of sedation for endoscopy .
although there are several drugs and techniques for the induction of sedation and reduction of pain during gastro - enteric endoscopies , there is no standardized method of sedation , and physicians themselves choose the best method regarding their experience .
benzodiazepines are mostly used for the goal of sedation , and among them , midazolam is the drug of choice .
opioids ( including pethidine and fentanyl ) , propofol , ketamine , and droperidol are other sedative drugs used for the same purpose .
ketamine could be used as a substitute of opioids and benzodiazepines for sedation during endoscopy with a wide spectrum effects on pain , amnesia , anesthesia , and sedation which can be administered intravenous , intramuscular , or even oral and rectal .
dose of 0.2 - 0.5 mg / body weight ( kg ) is usually used for reduction of pain and after 10 - 15 min , the full consciousness is returned .
delusion , auditory and visual hallucinations , and other side effects which may occurred in a period of 24 h after ketamine administration make some limitations for drug usage . a potential contractive effect on laryngeal muscles in unwell patients
previous studies had worked on the combination of ketamine and midazolam for the induction of sedation in pediatric gastro - enteric endoscopy but there is no similar study on adults .
in some areas of the world , ketamine is mostly used in sedation for endoscopy for all ages due to lack of other medications .
ketamine is also used for the reduction of neuropathic pains such as postherpetic neuralgia , complex regional pain syndrome , malignancy , orofacial pain , and limb phantom pain .
due to lack of enough data about the effect of ketamine on endoscopic sedation , we designed this study to evaluate the mentioned purpose .
this double - blinded , randomized clinical trial study was done by the grant of isfahan university of medical sciences during the years 2014 - 2015 with the group of patients who were candidates for gastro - enteric endoscopy .
inclusion criteria were the following : age between 18 and 65 years , absence of hypersensitivity or any contraindication for ketamine , absence of mental or physical retardation , and no history of hypertension , seizure , hyperthyroidism , immune deficiency , or increased intraocular pressure .
any patient who required emergent intervention or who showed drug sensitivity during the study was excluded from the study .
all the patients who had the inclusion criteria were informed about the study protocols and procedures and fulfilled a written consent , after that they were divided into two groups of case and controls by computer randomizing system meaning that every participant had a number categorized into case or control group by the computer .
a questionnaire including demographic data was fulfilled ; patients in case group administered 5 mg / body weight ( kg ) of ketamine , 30 min before the endoscopy and the control group received placebo in the same order . for complete blindness of the study ,
drug or placebo was solved in 10 cc of apple juice because of the bitter taste of ketamine and after that , medication was given to the patient by a nurse who was blinded about the study protocols .
after finishing the procedure , pain severity , degree of sedation , and physicians satisfaction of sedation were measured in both groups .
visual analog scale was used for pain assessment and physicians satisfaction of sedation was measured with the following scale : very bad ( scale 0 ) , bad ( scale 1 ) , intermediate ( scale 2 ) , good ( scale 3 ) , and very good ( scale 4 ) .
the sedation was also assessed with 1 = completely awake , 2 = awake but drowsy , 3 = asleep but responsive to verbal commands , 4 = asleep but responsive to tactile stimulus , and 5 = asleep and not responsive to any stimulus .
finally , all the gathered data were analyzed using spss-20 software ( spss inc . ,
chicago , illinois , usa ) ; t - test , independent t - test , mann - whitney test , and chi - square test were used for data analysis and p < 0.05 was considered statistically significant .
eighty - six patients ( 43 in case group and 43 in control group ) who were candidates for gastro - enteric endoscopy were included into this study ; of which 43 ( 50% ) individuals were males and 43 ( 50% ) were females .
as seen in table 1 , no differences were seen among groups regarding age , sex , and level of academic degree .
demographic and educational data of case and control group table 2 shows that the mean number of pain severity and discomfort during endoscopy were 2.4 1.8 and 5.81 1.48 in case and control groups , respectively , which showed significant differences among groups ( p < 0.001 ) [ figure 1 ] .
distribution of pain and discomfort , endoscopist 's satisfaction , sedation score , and retching in case and control groups severity of pain and nausea in case and control groups as seen in table 2 , none of the patients in the case group had severe nausea and pain otherwise there were five individuals in the control group who had severe nausea and pain .
overall , regarding categorization of pain and nausea in both groups , there was difference among groups .
endoscopist 's satisfaction measurement showed that most of the patients in the case group were categorized as completely satisfaction
( 32 patients ; 74.4% ) , but most of the patients in the control group were in relatively satisfaction group ( 13 patients ; 30.2% ) .
mann - whitney test revealed statistical difference among groups about physician 's satisfaction of sedation during endoscopy ( p < 0.001 ) [ figure 2 ] .
endoscopist 's satisfaction of sedation during endoscopy in case and control groups the sedation score was assessed in both groups , and patients who received ketamine had better sedative status ( mann - whitney test ; p < 0.001 ) .
none of the patients in the case group was completely awake , but all of the patients in the control group were awake .
the number of retching during endoscopy showed that individuals in the control group had more frequent retching episodes [ table 2 ] .
fifteen patients in the case group showed complications including nausea ( four patients ) , nausea and vomiting ( three patients ) , tachycardia ( six patients ) , and deep sedation ( one patient ) .
ketamine - induced sedation had more complications in comparison with those administered with placebo ( p < 0.001 ) .
the main goal of this study was to find a response to a question that is whether low - dose oral ketamine administration can ameliorate patients pain and discomfort and also increase physicians satisfaction during gastro - enteric endoscopy .
our data revealed that low - dose administration of ketamine , 30 min before initiation of the endoscopy , can significantly decrease patient 's pain and discomfort .
we also found that physicians reported better satisfaction of sedation in patients who received ketamine comparing with those who received placebo .
studies and experiences suggested that a successful gastro - enteric endoscopy can be done with a moderately potent sedative medication .
a previous meta - analysis revealed that a moderate sedation can increase patient and physician 's satisfaction .
although propofol is growing to be used as a moderate sedative medication , midazolam plus one opioid is considered as a standardized sedative method .
there are several studies worked on the effect of ketamine on pain reduction and most of them are focused on the intravenous injection of ketamine and its effect on some special pains such as neuropathic pains .
moharari et al . found that injection of 10 cc lidocaine plus 2 cc of ketamine into the urethra can significantly decrease pain during cystoscopy , especially in the first 5 min of the procedure .
another study designed to find a suitable sedative method for pediatric gastro - enteric endoscopy revealed that combination of oral ketamine with intravenous injection of midazolam is a perfect way to decrease pain and induction of sedation ; however , further episodes of vomiting were reported due to oral administration of ketamine .
78 pediatric patients showed that peri - tonsillar infiltration of ketamine ( 0.5 mg / kg ) leads to reduced pain and postsurgery vomiting after adenotonsillectomy .
another study on 180 patients aged 18 - 60 and candidates for cholecystectomy revealed that subcutaneous injection of 2 mg / kg of ketamine or intravenous injection of 1 mg / kg of ketamine 15 min before initiation of the surgery can decrease postsurgery pain .
combination of ketamine ( 0.25 mg / kg ) and meperidine ( 5 mg ) had been shown to have effects on pain reduction and decreased need to opioids after major abdominal surgeries in patients aged 15 - 60 years .
results of a recent investigation by shavakhi et al . revealed that sublingual administration of alprazolam resulted in better sedative status , decreased anxiety , and increased patient 's satisfaction during endoscopic procedures compared with oral route of administration .
lack of measurement of awaking time and ending time of procedure can be mentioned as our study limitation .
as we searched , there was no study which compared the effect of ketamine compared with placebo , so we did not compared the effect of ketamine with other sedative agents .
our data showed that a low - dose oral administration of ketamine could make a suitable sedation for gastro - enteric endoscopy .
patients experienced less pain and less frequent retching and physicians had greater satisfaction of sedation compared with those placebo - treated group .
we would like to suggest using ketamine as a suitable medication for the induction of sedation during gastro - endoscopic procedures if there is no contraindication .
sm contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
ak contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
mk contributed in the conception and design of the work , drafting and revising the draft , and agreed for all aspects of the work .
sd contributed in the drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
pa contributed in the conception and design of the work , drafting and revising the draft , and agreed for all aspects of the work .
mm and rk contributed in the conception and design of the work and agreed for all aspects of the work . | background : a suitable sedative status during gastro - enteric endoscopies results in better physicians approach and more stable view of internal organs .
therefore , we evaluated the effect of ketamine for sedation in endoscopic procedures of adult patients.materials and methods : patients who were candidates for gastro - enteric endoscopy during the years 2014 - 2015 were included into the study and divided into two groups of case ( administered 5 mg / kg of oral ketamine half an hour before initiation of the procedure ) and control ( administered placebo in a same pattern ) .
after endoscopy , patients and physicians satisfaction of sedation was assessed .
spss-22 was used for data analysis.results:eighty-six patients participated into the study of which divided into each groups .
the pain and discomfort scores were 2.4 1.8 and 5.81 1.48 in case and control groups , respectively , ( p < 0.001 ) .
mann - whitney test revealed statistical difference among groups about physician 's satisfaction of sedation during endoscopy ( p < 0.001 ) .
patients who received ketamine had better sedative status ( p < 0.001 ) .
none of the patients in the case group was completely awake but all of the patients in the control group were awake .
the number of retching during endoscopy showed that individuals in the control group had more frequent retching episodes ( p = 0.04).conclusion : low - dose oral administration of ketamine could make a satisfied sedation for gastro - enteric endoscopy . |
the finnish type 1 diabetes prediction and prevention ( dipp ) study was carried out in three university hospitals ( turku , oulu , and tampere ) .
more than 90% of the 11,000 babies born annually in these centers take part in cord blood screening for type 1 diabetes associated hla genotypes ( 7 ) .
infants carrying the high - risk genotype ( hla dqb1 * 02/0302 ) or the moderate - risk genotypes ( hla dqb1 * 0302/x ; x*02 , * 0301 , * 0602 , or * 0603 ) are eligible for a prospective follow - up study in which participants are monitored for the appearance of daas and type 1 diabetes ( supplementary fig . 1 [ available at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1305/dc1 ] ) ( 8,9 ) .
information on the family history of type 1 diabetes is collected with structured questionnaires completed by the parents soon after the birth of the baby . in the study centers in oulu and tampere ,
the clinical follow - up visits take place at the age of 3 , 6 , 12 , 18 , and 24 months and , after that , annually . in turku ,
the visits occur every 3 months until the age of 2 years and subsequently every 6 months , which makes it theoretically possible that children from turku could seroconvert at a younger age than other dipp children , but according to current analyses this was not the case . for the seroconverted subjects ,
the follow - up visits are organized every 3 months . at the follow - up visits ,
venous blood samples are obtained , and for the current analyses , icas were used as the primary screening tool for -cell autoimmunity .
subjects with transplacentally transferred maternal antibodies were regarded as seronegative as long as no de novo synthesis of antibodies was observed ( 10 ) .
persistent autoantibody positivity ( prefix p ) was defined as positivity in at least two sequential samples , the last sample available being positive . the last pre - diabetic and/or the first diabetic samples were taken into account when defining the persistence of the autoantibody status . participants with persistent positivity for at least two of the autoantibodies analyzed were eligible for a randomized , double - blinded , and placebo - controlled intervention trial with nasally administrated insulin .
codes for the intervention treatment were opened in november 2007 , and the results showed that the intervention had no effect on the progression rate to clinical diabetes ( 11 ) .
the present study cohort comprised dipp children who had remained in the follow - up study for at least 1 year or presented with diabetes before the age of 1 year ( one case diagnosed at the age of 0.9 years ) by the end of august 2004 .
the cohort included 7,410 children ( 3,897 male subjects [ 52.6% ] ) , and 177 ( 2.4% ) had a family member affected by type 1 diabetes at the time of birth .
the closing time point for the data on autoantibodies and progression to type 1 diabetes was 31 december 2008 .
screening for hla dqb1 genotypes was performed on cord blood samples by time - resolved triple - label hybridization ( 12 ) .
autoantibodies were measured on serum samples : islet cell autoantibodies ( icas ) with immunofluorescence ( 13 ) and antibodies against insulin ( iaas ) , antibodies against gad ( gadas ) , and islet antigen 2 ( ia-2a ) with specific radiobinding assays ( 1416 ) .
the cut - off values for ica , iaa , gada , and ia-2a positivity were 2.5 juvenile diabetes foundation units ( jdfu ) , 3.48 relative units ( ru ) , 14.13 world health organization ( who ) units / ml (= 5.36 ru ) , and 1.91 who units / ml (= 0.43 ru ) , respectively .
the reference values for the iaa , gada , and ia-2a assays were based on the 99th percentile of > 370 nondiabetic finnish children and adolescents .
samples with iaa , gada , or ia-2a values between the 97th and 99.5th percentiles , as well as all the ica - positive samples , were retested to confirm the antibody status . the disease sensitivity and specificity of the ica assay were 100 and 98% , respectively , based on an international standardization workshop round ( 17 ) . according to the 2005 diabetes autoantibody standardization program ( dasp ) workshop ,
the disease sensitivity of the iaa , gada , and ia-2a assays were 58 , 82 , and 72% , respectively , while corresponding specificities were 98 , 96 , and 100% , respectively .
the diagnosis of type 1 diabetes was based on who criteria ( 18 ) and the primary case ascertainment done by reviewing the local registers of children with newly diagnosed type 1 diabetes in each of the three participating university hospitals .
the finnish pediatric diabetes register was used as the secondary source of ascertainment ( 19 ) .
the local ethics committees approved the protocol of the dipp study , and written informed consent was obtained from the legal representatives of the participating children . to analyze the predictive characteristics of ica - based autoantibody combinations ,
each child could belong to one group only , except when the categories two or more and three or more daas were analyzed . for subjects with fluctuating autoantibodies , the maximal combination or , in the case of various combinations with similar numbers of positive autoantibodies , the first combination to appear
disease sensitivity and specificity , as well as positive ( ppv ) and negative ( npv ) predictive values and likelihood ratios ( + lr and lr ) , were determined for ica alone and for ica in combination with the other three autoantibodies . the kaplan - meier method with the log - rank test
distributions between groups were tested with the test and correlations analyzed with the pearson 's ( r ) or spearman 's methods ( rs ) .
cis were given at 95% ( 95% ci ) , and statistical significance was set at p < 0.05 ( two - tailed ) .
statistical analyses were performed with spss 16.0 for windows ( spss , chicago , il ) .
to analyze the predictive characteristics of ica - based autoantibody combinations , participants were categorized by their maximal autoantibody status by the end of december 2008 .
each child could belong to one group only , except when the categories two or more and three or more daas were analyzed . for subjects with fluctuating autoantibodies , the maximal combination or , in the case of various combinations with similar numbers of positive autoantibodies , the first combination to appear
disease sensitivity and specificity , as well as positive ( ppv ) and negative ( npv ) predictive values and likelihood ratios ( + lr and lr ) , were determined for ica alone and for ica in combination with the other three autoantibodies .
the kaplan - meier method with the log - rank test was used to analyze and compare the cumulative diabetes risks .
odds ratios ( ors ) for the risk of type 1 diabetes were calculated . distributions between groups were tested with the test and correlations analyzed with the pearson 's ( r ) or spearman 's methods ( rs ) .
cis were given at 95% ( 95% ci ) , and statistical significance was set at p < 0.05 ( two - tailed ) .
statistical analyses were performed with spss 16.0 for windows ( spss , chicago , il ) .
we observed from birth 7,410 children for positivity for daas and progression to type 1 diabetes over a median follow - up time of 9.2 years ( range 0.914.2 ) .
the median follow - up time for subjects remaining unaffected by type 1 diabetes was 9.3 years ( 5.414.2 ) , and the median age at diagnosis among 180 progressors ( 93 male subjects [ 2.4% ] ) was 5.0 years ( 0.912.5 ) .
the median age at the initial seroconversion was 4.2 years ( 0.213.7 ) among unaffected ica - positive subjects , whereas among progressors , it was 1.5 years ( 0.39.6 ; p < 0.001 ) .
progressors reached the maximal ica - based autoantibody status by the median age of 2.2 years ( 0.510.1 ) , while in unaffected subjects , the maximal autoantibody status was observed at the age of 5.1 years ( 0.513.7 ; p < 0.001 ) .
delay from the initial seroconversion to maximal autoantibody positivity varied between 0.0 and 11.0 years , and among progressors and unaffected subjects , the median delays were 0.5 and 0.0 years ( p < 0.001 ) , respectively .
the high - risk genotype ( dqb1 * 02/0302 ) was carried by 1,575 children ( 21.3% ) and the moderate - risk genotypes ( dqb1 * 0302/x ; x * 02 or a protective allele ) by 5,835 children ( 78.7% ) .
the high - risk genotype was associated with higher risks for developing -cell autoimmunity , positivity for multiple autoantibodies , persistent positivity , and type 1 diabetes ( p < 0.001 for all comparisons ) ( supplementary table 1 ) .
the corresponding ors for the high- versus moderate - risk groups were 1.7 ( 95% ci 1.42.0 ) , 2.4 ( 1.93.1 ) , 1.7 ( 1.42.0 ) , and 3.2 ( 2.14.8 ) , respectively .
altogether , 1,173 subjects ( 15.8% ) , 155 of whom were progressors , tested positive for icas during the follow - up . a majority ( n = 967 ; 82.4% ) of the first autoantibody - positive samples were ica positive , while positivity for either icas or iaas was seen in 93.3% ( 1,094 of 1,173 ) of the initially positive samples .
seventeen ( 68.0% ) of 25 progressors who had remained ica - negative during the pre - diabetic phase were positive for iaas either before or at the time of the diagnosis ( table 1 ) , and , in all , among the ica - positive children , those who tested positive also for iaas had a higher cumulative disease risk ( 59.6% [ 95% ci 49.969.3 ] ) than those who remained iaa negative ( 10.8% [ 6.814.9 ] ; p < 0.001 ) .
twelve of 15 ( 80% ) progressors without any signs of pre - diabetic -cell autoimmunity did not adhere to the follow - up schedule of the dipp study . among these subjects ,
the median delay from the last sampling to diagnosis was 3.8 years ( range 1.96.2 ) .
all previously seronegative progressors having samples available at diagnosis had developed -cell autoimmunity by that time , and all but one tested positive for multiple autoantibodies .
progressors remaining ica negative during the pre - diabetic phase x , nonprotective hla allele .
the age at which subjects seroconverted had a predictive role regarding the risk of type 1 diabetes . among all ica - positive subjects ,
those with seroconversion before the age of 2 years had the highest cumulative disease risk ( 36.9% [ 95% ci 28.545.3 ] ) ( fig .
1a ) , and for that age - group , the or for type 1 diabetes was 5.0 ( 95% ci 3.57.1 ) when compared with those who had seroconverted after the age of 2 years .
subjects seroconverting under the age of 2 years were also more often positive for multiple autoantibodies at first positive sampling ( 18.3 vs. 12.1% in subjects with seroconversion at or after the age of 2 years , p = 0.006 ) .
the median delay from the initial seroconversion to diagnosis was 2.8 years ( range 0.0210.9 ) among the ica - positive progressors , and this delay did not correlate with the age at seroconversion ( rs = 0.005 , p = 0.95 ) .
effect of the seroconversion age on the diabetes - free survival ( a ) and progression to type 1 diabetes in relation to initial ica titer ( jdfu ) ( b ) .
the median ica level in the first ica - positive samples was 5 jdfu ( range 3640 ) among nonprogressors and 15 jdfu ( 4668 ) ( p < 0.001 ) in progressors .
the higher the initial ica value , the higher the cumulative disease risk ( fig .
the 5-year progression rate for those with a low initial ica level ( < 10 jdfu ) was 5.7% ( 95% ci 3.97.5 ) , while the corresponding values for those with moderate ( 1019 jdfu ) and high ( 20 jdfu ) ica titers were 31.8% ( 21.841.8 ) and 61.2% ( 51.171.9 ) , respectively . during the follow - up ,
the difference in ica levels became more prominent between progressors and nonprogressors : the peak ica titer among progressors reached 168 jdfu ( range 52,620 ) , while it remained at 5 jdfu ( 32,620 ) in nonprogressors ( p < 0.001 ) .
prospective observations from the time point at which the maximal ica level was observed showed a 5-year cumulative disease risk of 2.3% ( 95% ci 0.34.3 ) among those with a low ica ( < 10 jdfu ) level at that time , whereas among those with moderate ( 1019 jdfu ) and high ( 20 jdfu ) ica levels , the risk estimates were 11.7% ( 2.920.5 ) and 76.5% ( 61.491.6 ) ( p < 0.001 between all groups ) , respectively . the maximal ica level correlated clearly with the number of detectable autoantibodies at sampling ( rs = 0.68 , p < 0.001 ) , but the correlation between the ica titer and type 1 diabetes remained significant , even after correcting for the number of positive autoantibodies ( rs = 0.10 , p < 0.001 ) . to further analyze the predictive role of icas in combination with the other three autoantibodies ,
frequencies and predictive characteristics ( sensitivity , specificity , ppv , npv , + lr , lr , and cumulative disease risks ) of the autoantibody combinations are presented in table 2 and figs .
positivity for four antibodies was associated with the highest disease sensitivity ( 54.4% ) and npv ( 98.9% ) and the lowest lr ( 0.5 ) .
the combination of persistent ica and iaa positivity resulted in the highest ppv ( 91.7% ) , + lr ( 441.8 ) , cumulative disease risk ( 100% ) , and specificity ( 100% ) .
especially , the combination of icas and gadas resulted in a low progression rate , thus decreasing also the risk estimate of double positivity .
transient and persistent ica positivity had different predictive characters , since only 1.7% ( 95% ci 04.0 ) of those with transient ica - based positivity developed type 1 diabetes during follow - up , whereas among those with ( at least ) pica positivity , the proportion was 41.2% ( 34.148.3 ) ( p
the difference between the seronegative and transiently ica - positive subjects remained small but significant ( 0.5% [ 95% ci 0.30.7 ] vs. 1.7% ) ( p = 0.008 ) .
the pica - based combinations of multiple persistently positive autoantibodies had highly variable disease risks , the highest associating with the combination of picas and piaas ( 100% ) and the lowest with picas and pgadas ( 11.2% ) .
persistent iaa positivity seemed to distinguish those with a high disease risk and rapid progression to type 1 diabetes from those with a lower disease risk and slower progression rate . in this population , the piaa positivity
associated 5-year disease risk was 70.7% ( 95% ci 62.279.1 ) compared with that of 11.9% ( 8.415.4 ) observed among the ica - positive subjects lacking piaa positivity . since all children had been observed for at least 5 years , the 5-year predictive characteristics are presented in supplementary table 2 .
the predictive characteristics in relation to hla genotype are shown in supplementary table 3 , while the corresponding characteristics of those 7,077 children who had no affected first - degree relative are presented in supplementary table 4 .
predictive characteristics ( sensitivity , specificity , ppv , npv , + lr , lr , and cumulative disease risk ) of the four diabetes - associated autoantibodies ( daas , icas , iaas , and gadas , and ia-2a ) data are % ( 95% ci ) unless otherwise indicated .
type 1 diabetes / all progressors / all subjects positive for the given specificity ; p , persistently positive autoantibody . in the case of defined autoantibody categories ,
* ica positivity - based categorization . the data in bold represent the highest or lowest ( lr ) value for each characteristic . progression to type 1 diabetes ( t1d ) in relation to number of positive autoantibodies ( a ) and
combinations of double ( b ) and triple ( c ) positivity further defined . *
p < 0.001 ; p = 0.02 ; p = 0.04 . progression to type 1 diabetes ( t1d ) in relation to number of persistently positive autoantibodies ( a ) and combinations of persistent double ( b ) and triple ( c ) positivity further defined .
the virtues of the present work lie in the extensive series of children with hla - conferred diabetes susceptibility derived from a background population with the highest incidence of type 1 diabetes in the world , and the fact that they were observed already from birth for the appearance of signs of -cell autoimmunity and progression to overt type 1 diabetes . because of the finnish pediatric diabetes register ( 19 ) , it was also possible to trace the majority of progressors that had dropped out from the regular pre - diabetic follow - up and often to even get a blood sample for autoantibody analyses at the time of their diagnosis .
our main limitation was using icas as the only primary screening tool for -cell autoimmunity , thus missing some ica - negative subjects with positivity for molecular autoantibodies .
the rationale for this approach was based on the knowledge available in 1994 at the initiation phase of the dipp study . at that time
, ica represented the autoantibody reactivity with the most robust information on their predictive value ( 20,21 ) .
the sensitivity of the current screening program would have increased from the observed 86% for icas to 97% , if iaas had been added to the initial screening .
this would have reduced the number of pre - diabetically seronegative progressors . according to our experience , however , the main reason for pre - diabetic seronegativity was discontinuation of the regular follow - up and , extremely rarely , the fact that no seroconversion had occurred . in the present series
, there were three progressors having otherwise clinically obvious type 1 diabetes but no known detectable autoantibodies at the last follow - up visit 48 months before the diagnosis .
unfortunately , for one of these individuals , no autoantibody sample was obtained at the diagnosis of clinical disease , while the other two had developed at least ica and iaa positivity by the time of their diagnosis .
twelve of 15 prediabetically seronegative subjects had dropped out from the regular follow - up , and in this group , the shortest delay from the last pre - diabetic sample to diagnosis was 1.9 years . with this observation in mind
, one might suggest that in any screening program based on autoantibody detection in young children , the sampling interval should not exceed 2 years . in the general childhood population selected for disease risk
related hla genotypes , isolated low - level ica positivity did not confer a significant increase in the disease risk when compared with autoantibody - negative children , but the risk of type 1 diabetes associated with this marker was related to its level as well as with multipositivity .
these findings are similar to those observed among first - degree relatives of children with type 1 diabetes ( 2224 ) . however , the strong correlation between the ica titer and the number of positive molecular autoantibodies indicated the latter phenomenon to be the true risk marker rather than the ica titer . as previously reported among first - degree relatives of patients with type 1 diabetes
, we also observed that the younger the child at the seroconversion , the higher the risk of presenting with clinical diabetes during the observation period ( 25 ) .
part of this finding may result from the variation in the follow - up times among the subjects , since the dipp study is still ongoing , but according to the analysis of diabetes - free survival , the group of the youngest seroconverters differed from the older ones already 2 years after the seroconversion ( progression rate 9.7 vs. 1.43.3% in the older age - groups ) .
the degree of the variation in sensitivity , ppv , + lr , and cumulative disease risk estimates associated with the different combinations of multipositivity was conspicuously wide .
sensitivity of the categorized autoantibody combinations remained mainly modest , except for the quadruple positivity with a slightly higher value of 54% .
specificity was > 98% for all other markers except isolated ica and pica positivity , and the npv values were all > 97% . remarkably high ppv ( 92% ) , +
lr ( 442 ) , and disease risk ( 100% ) were associated with the combination of persistently positive icas and iaas .
all persistent autoantibody combinations except the combination of picas and pgadas had + lr values > 10 , indicating an increased disease risk ( 26 ) .
the predictive characteristics of the combined ica and gada positivity resembled single ica positivity , and since some of the ica positivity is derived from gada reactivity , one may assume that the findings regarding this combination may be at least partly explained by overlapping antibody reactivity .
observations from the current study confirm that in the general population the combination of hla genotyping and autoantibody detection can provide predictive values similar to those reported among first - degree relatives of affected patients ( 2729 ) .
for example , in the childhood diabetes in finland ( dime ) study ( 29 ) , among siblings of children with newly diagnosed type 1 diabetes , positivity for at least three autoantibodies was associated with a 5-year cumulative risk of 57% , while the corresponding risk was 62% in the present series .
positivity for multiple autoantibodies is , however , probably less frequent in the background population than among family members , even after screening for individuals with hla - conferred disease susceptibility . in this study ,
the frequency of positivity for three or more daas was 3.5% ( 263 of 7,410 ) , while the corresponding figure for dime siblings was 4.6% .
accordingly , to identify similar numbers of individuals at high risk ( > 50% over 5 years ) of disease progression from the background population with hla - defined diabetes susceptibility , and from siblings of affected children , one - third more children should be screened from the background population .
nevertheless , persistently multipositive children in the general population represent the majority of individuals at risk for type 1 diabetes , and without preventive measures covering this part of the population , only a minor proportion of future cases can be prevented .
given that the natural progression rate is extremely high in this group of children , especially among those developing persistent positivity for iaas , these children might represent a subgroup in which more aggressive preventive treatments may be justified in the future .
altogether , our experience shows that it is feasible to observe children with hla - defined diabetes susceptibility from birth and to identify individuals developing -cell autoimmunity among them .
as soon as effective preventive treatments are available , prevention programs based on hla genotyping and regular autoantibody analyses may well be relevant in high - incidence countries , such as finland .
| objectiveas data on the predictive characteristics of diabetes - associated autoantibodies for type 1 diabetes in the general population are scarce , we assessed the predictive performance of islet cell autoantibodies ( icas ) in combination with autoantibodies against insulin ( iaas ) , autoantibodies against gad , and/or islet antigen 2 for type 1 diabetes in children with hla - defined disease predisposition recruited from the general population.research design and methodswe observed 7,410 children from birth ( median 9.2 years ) for -cell autoimmunity and diabetes . if a child developed ica positivity or diabetes , the three other antibodies were measured in all samples available from that individual .
persistent autoantibody positivity was defined as continued positivity in at least two sequential samples including the last available sample.resultspre-diabetic ica positivity was observed in 1,173 subjects ( 15.8% ) , 155 of whom developed type 1 diabetes . with ica screening , 86% of 180 progressors
( median age at diagnosis 5.0 years ) were identified .
positivity for four antibodies was associated with the highest disease sensitivity ( 54.4% ) and negative predictive values ( 98.3% ) and the lowest negative likelihood ratio ( 0.5 ) .
the combination of persistent ica and iaa positivity resulted in the highest positive predictive value ( 91.7% ) , positive likelihood ratio ( 441.8 ) , cumulative disease risk ( 100% ) , and specificity ( 100% ) .
young age at seroconversion , high ica level , multipositivity , and persistent positivity for iaa were significant risk markers for type 1 diabetes.conclusionswithin the general population , the combination of hla and autoantibody screening resulted in disease risks that are likely to be as high as those reported among autoantibody - positive siblings of children with type 1 diabetes . |
face processing is a visual skill that has received a great deal of research attention over the past several decades .
one reason for this is because of the unique perceptual skills that are involved in face processing : for example , face perception relies on processing mechanisms that are much more sensitive to image inversion ( e.g. , freire , lee , & symons , 2000 ; rossion & gauthier , 2002 ; yin , 1969 ) and configuration ( e.g. , hole , 1994 ; maurer , legrand , & mondloch , 2002 ; young , hellawell , & hay , 1987 ) than are other forms of object perception .
another reason for this research attention is because of the well - established role of specific cortical regions in face processing .
neurons in both the fusiform face area ( ffa ; kanwisher , mcdermott , & chun , 1997 ; sergent & signoret , 1992 ) and more recently the occipital face area ( ofa ; gauthier et al . , 2000 ; rossion et al . , 2003 ; steeves et al . ,
2005 ) have been demonstrated to play important roles in the mediation of face - specific behaviors . given the volume of research into both the perception and neuroimaging of face - specific behaviors
, researchers are now tasked with determining the relationship between neurons and behavior in face processing .
one of the main goals of ongoing research is to try and determine which aspects specific of face processing are mediated by the identified neural structures .
one facial characteristic that has been investigated extensively is the bilateral symmetry of the face .
facial symmetry plays an important role in a variety of different face perception skills , including judgments of facial attractiveness ( e.g. , grammer & thornhill , 1994 ; scheib , gangestad , & thornhill , 1999 ) , and social attention ( e.g. , langton , 2000 ) .
recent evidence suggests that the mechanisms involved in processing face symmetry are functionally different from those mechanisms involved in other lower level forms of symmetry processing .
for example , rhodes , peters , lee , morrone , and burr ( 2005 ) demonstrated that symmetry detection is reduced in inverted and contrast - reversed faces , which are manipulations that are known to impair face identification .
moreover , the ability to detect symmetry is reduced for bandpass filtered faces , whereas the mechanisms involved in lower - level symmetry detection are not sensitive to spatial scale ( dakin & hess , 1997 ; rainville & kingdom , 2002 ) . together , these results suggest that face symmetry is a feature that is processed by higher level mechanisms than those involved in basic symmetry perception .
little and jones ( 2006 ) further demonstrated that while preference ratings for inverted symmetric faces were lower relative to ratings for upright symmetric faces , the ability to detect symmetry in both upright and inverted faces was not significantly different .
they suggest that this demonstrates that the mechanisms involved in detecting facial symmetry may play a more important role in aesthetic face judgments than they do in face detection and recognition .
if this is true , then symmetry may not be an important feature in tasks that probe the ability to detect changes in face identity . in both of these studies , the stimuli that were used were realistic face images , where symmetry was achieved by either averaging the face with its mirror reversal ( rhodes , peters , & ewing , 2007 ; rhodes et al . , 2005 ) or by manually repositioning the facial features to symmetric coordinates ( little & jones , 2006 ) .
these symmetrization techniques both result in realistic face stimuli that possess features that are geometrically symmetric , and performance for determining whether or not subjects can discriminate a symmetric face from its asymmetric counterpart can be measured . however , in neither of these studies was the strength of symmetry ( or asymmetry ) manipulated .
another method of evaluating sensitivity to facial asymmetry is to manipulate the degree of symmetry and evaluate thresholds for symmetry detection .
threshold , in this instance , would refer to the minimum amount of geometric variation that would need to be added to one side of the face for observers to reliably detect asymmetry .
one advantage for using a quantifiable metric for symmetry manipulation is that asymmetry thresholds can then be directly compared to the same metric thresholds for discriminating the identity of the face . in other words , thresholds for detecting facial asymmetry and facial identity
can be directly compared in a meaningful way . in the current set of studies , we evaluated sensitivity to facial asymmetry using a set of synthetic faces where the geometric identity of the face can be precisely controlled .
these faces retain the geometric properties of individual faces , but eliminate other cues that can be used to judge facial identity , such as shading and texture ( wilson , loffler , & wilkinson , 2002 ) .
previous research has demonstrated that these highly reduced stimuli capture the identities of individual faces ( wilson et al . , 2002 ) , and elicit bold responses in the ffa that are similar to the bold responses elicited by photographs of faces ( loffler , yourganov , wilkinson , & wilson , 2005 ) .
these results suggest that the synthetic faces recruit the same neural mechanisms used to process photorealistic faces .
we assessed sensitivity to facial asymmetry by selectively adding individual geometric identity to only one half of the face , and measuring the minimum amount of asymmetry that was required for subjects to reliably determine whether or not the face was asymmetric .
as this form of geometric manipulation can also be used to measure thresholds for identifying an individual face ( wilson et al . , 2002 ) , we could also directly compare sensitivity thresholds for discriminating asymmetry and identity in faces .
we found that subjects were 2 times more sensitive to facial asymmetry , even though only half of the geometric information was added to the whole face . when the same comparison between sensitivity for shape asymmetry and identity was measured using a set of nonface geometric shapes ( i.e. , radial frequency ( rf ) patterns ) , the opposite pattern of responses was observed , suggesting that the advantage for detecting asymmetry over shape is specific to face stimuli .
this exquisite sensitivity to facial asymmetry was not affected by the inversion of the faces , nor was it different for faces that were geometrically constructed from mean or nonmean faces ( a manipulation that does influence identity thresholds ) . taken together , we believe that these results suggest that facial asymmetry is processed by face - specific mechanisms that differ from the mechanisms that are involved in face identification .
to evaluate both identity and asymmetry sensitivity , we used the synthetic faces described by wilson et al ( 2002 ) .
these faces are generated by identifying 16 equiangular landmarks on the outer head contour , and nine landmarks on the hairline , on digital photographs of 40 individual faces .
these landmarks are used to regenerate the hair and head face contours through the sum of seven radial frequencies ( wilkinson , wilson , & habak , 1998 ) .
the representation of the outer and inner head contour ( rhead ) in polar coordinates is thus :
( 1)rhead = rmeann=17ancos(2n)+bnsin(2n ) ,
where an and bn are amplitudes defining head shape ( n = 17 ) and are defined relative to the mean head radius ( rmean ) .
generic internal face features are also placed according to 12 additional landmarks ( with two xy coordinates for the placement of each eye ) determined from the individual faces .
each individual face is therefore described as a 37 element vector describing the face coordinates .
the resultant faces are bandpass filtered with a 2.0 octave difference of gaussians filter with a peak frequency of 10 cycles per face , as this is the range of spatial frequencies that contains the most relevant information for face identification ( gold , bennett , & sekuler , 1999 ; nsnen , 1999 ) . to generate synthetic faces where the individual identity of the face can be continuously varied , the 37 element vectors describing the 40 individual faces are averaged together to determine the mean face coordinates .
this mean vector is then subtracted from each of the individual identity vectors , yielding a difference vector that describes the individual variation from the mean . to control the amount of geometric identity within the face
, the amplitudes that define the identity of the individual face are added to the amplitudes of the mean face for strengths ranging from 0 to 15% mean head radius .
the identity strength is therefore described as the percent variation of the individual face vector from the mean .
for a more comprehensive description of the creation of these synthetic faces , see wilson et al .
an example of an identity continuum for one individual face with six identity strengths ranging from 0 to 15% is presented in figure 1a .
the percentages given below each image provide the amount of geometric variation from the mean head .
, the geometric variation has been added to the left half of the face , while the right side retains the mean head geometry .
the percentage given below refers to the geometric variation on the asymmetric side of the face .
asymmetric faces were created by fusing one side of a face with varying amounts of facial identity to the complimentary side of the mean ( i.e. , 0% identity ) face . to ensure that there were no artifacts due to contour misalignments between the two face halves , a weighted average of the gray levels 30 pixels from the vertical midline in each half was calculated to create a smooth contour .
this fusion was completed for six identity strengths ranging from 0 to 10% for each individual , and also for the left and right sides of the individual face .
an example of one asymmetric face trajectory is presented in figure 1b , where the asymmetry is added to the left half of the face .
the percentages provided below each exemplar correspond to the variation from the mean head radius in the asymmetric half of the face .
the rf patterns that were used as a nonface shape control stimulus were generated as described by wilkinson et al .
the shapes are generated by applying a sinusoidal modulation about a mean circle with a cross - sectional luminance profile of a fourth derivative gaussian ( figure 4a , top ) .
the frequency of the modulation determines the number of lobes in the shape , and the amplitude determines the strength of the shape and is expressed as the percent variation from the mean shape ( i.e. , circular ) radius . in the current study , we generated rf patterns at two phases ( 90 and 270 ) with a rf of 8 , which is well above the frequency for optimal performance ( wilkinson et al . , 1998 ) .
for each phase , we generated six shapes with shape strengths ranging from 0 to 1.5% mean shape radius .
to create asymmetrical rf patterns , we used the same technique that was used to create asymmetrical faces .
rf patterns were bisected along the vertical midline , and were fused to the complimentary side of the mean circle .
again , the contour between each side was smoothed by taking the weighted average of the middle 30 pixels .
an example of the asymmetric rf pattern is provided in the bottom of figure 4a .
two sets of experiments were conducted to determine whether the sensitivity to facial asymmetry was affected by stimulus manipulations that have been demonstrated to affect face identification .
first , asymmetry thresholds were evaluated with faces that were inverted 180 ( figure 5a ) .
all other aspects of the stimuli in this condition were identical to the upright faces used in the primary experiment .
second , asymmetry thresholds were evaluated for faces that were constructed about a nonmean face . in these stimuli , the amplitudes that define the identity of an individual face were added to a face with 12% identity variation from the mean .
increasing asymmetry was then added to the left or right side of the nonmean face in the same manner as it was in the faces constructed about the mean .
an example of an asymmetric face constructed about a nonmean face is provided in figure 6b .
individual thresholds for discriminating radial frequency ( rf ) patterns in both identity and asymmetry conditions .
subjects were required to either discriminate a full rf8 pattern from a mean circle ( top ( a ) : identity condition ) , or a pattern where the rf8 contour was only added to one half of the shape ( bottom ( a ) : asymmetry condition ) .
( b ) identity ( left ) and asymmetry ( right ) thresholds for rf patterns for five individual subjects .
two subjects ( lh and am ) had equivalent thresholds for each condition , whereas the other three subjects had lower thresholds for the identity condition than for the asymmetry condition .
( a ) thresholds for detecting asymmetry were compared when the faces were presented upright ( top ( a ) ) , or when rotated 180 ( bottom ( a ) ) .
( b ) individual asymmetry thresholds for three subjects for the upright ( left ) and inverted ( right ) faces .
asymmetry thresholds for faces constructed using either the mean or a nonmean face as a geometric base .
( a ) asymmetric faces were created by adding geometric asymmetry to either the mean face ( top ( a ) ) , or a nonmean face possessing 10% geometric identity ( bottom ( a ) ) .
the percentages given below refer to the amount asymmetry added to the face . in both examples ,
( b ) individual asymmetry thresholds for three subjects for the mean ( left ) and nonmean ( right ) base faces .
stimuli were presented on an intel mac mini with a 1.66 ghz core duo processor and a 17 inch seanix monitor with a resolution of 1024 1280 and a 75 hz refresh rate .
stimulus generation and presentation was accomplished using psychtoolbox functions ( brainard , 1997 ) and custom software developed in the matlab programming environment .
subjects viewed the stimuli from a distance of 57 cm , which was maintained by the use of a chin rest .
thresholds were determined using a two interval - forced choice procedure , where one interval contained either a face from the identity trajectory for one individual ( identity condition ) or a face from the asymmetric trajectory ( asymmetry condition ) , and the other interval contained the mean or symmetric face .
subjects responded , by keyboard , which interval contained the individual or asymmetric face . to ensure that discriminations were not based on local changes in contour features , the size of the faces across the two presentation intervals was randomly jittered from between 2.3 2.9 and 4.6 5.8. each face was presented for 120 ms , followed by a noise mask presented for 200 ms .
the noise mask was matched to the size of the preceding face , and was bandpass filtered to contain the same peak spatial frequency as the faces . within each experimental run
four individual identity trajectories were created , with identity strengths ranging from 0 to 15% mean head radius in 3% increments . in the asymmetry condition ,
two individual identities were used ; however , for each identity , there were two asymmetry ( i.e. , left and right ) conditions resulting in four asymmetry trajectories in each experimental run .
thresholds did not differ between the left and right asymmetry conditions , and data across these conditions were collapsed into simply the asymmetry condition .
therefore , in each experimental run , there were 24 total face stimuli ( four trajectories six increments ) , which were each presented 20 times for a total of 480 trials .
percent correct data for the 6 identity strength increments ( x ) were fit with a quick ( 1974 ) function of the form :
( 2)f(x)=10.52(xa)
where corresponds to threshold ( 75% correct ) and reflects the slope of the function .
all of the data presented reflect the average threshold estimation for a minimum of three experimental runs for that condition , where each of the different runs contains faces taken from different identity trajectories .
although statistical significance was determined and presented using standard parametric statistics , significant effects were confirmed using non - parametric analyses ( not presented ) .
five observers participated in the experiments evaluating face and rf asymmetry thresholds , whereas three observers participated in the conditions evaluating asymmetry thresholds with inverted and nonmean faces .
the authors ( na and cg ) participated in all of the conditions . all other observers were nave to the purpose of the experiments .
to evaluate both identity and asymmetry sensitivity , we used the synthetic faces described by wilson et al ( 2002 ) .
these faces are generated by identifying 16 equiangular landmarks on the outer head contour , and nine landmarks on the hairline , on digital photographs of 40 individual faces .
these landmarks are used to regenerate the hair and head face contours through the sum of seven radial frequencies ( wilkinson , wilson , & habak , 1998 ) .
the representation of the outer and inner head contour ( rhead ) in polar coordinates is thus :
( 1)rhead = rmeann=17ancos(2n)+bnsin(2n ) ,
where an and bn are amplitudes defining head shape ( n = 17 ) and are defined relative to the mean head radius ( rmean ) .
generic internal face features are also placed according to 12 additional landmarks ( with two xy coordinates for the placement of each eye ) determined from the individual faces .
each individual face is therefore described as a 37 element vector describing the face coordinates .
the resultant faces are bandpass filtered with a 2.0 octave difference of gaussians filter with a peak frequency of 10 cycles per face , as this is the range of spatial frequencies that contains the most relevant information for face identification ( gold , bennett , & sekuler , 1999 ; nsnen , 1999 ) . to generate synthetic faces where the individual identity of the face can be continuously varied , the 37 element vectors describing the 40 individual faces are averaged together to determine the mean face coordinates .
this mean vector is then subtracted from each of the individual identity vectors , yielding a difference vector that describes the individual variation from the mean . to control the amount of geometric identity within the face
, the amplitudes that define the identity of the individual face are added to the amplitudes of the mean face for strengths ranging from 0 to 15% mean head radius .
the identity strength is therefore described as the percent variation of the individual face vector from the mean .
for a more comprehensive description of the creation of these synthetic faces , see wilson et al .
an example of an identity continuum for one individual face with six identity strengths ranging from 0 to 15% is presented in figure 1a .
the percentages given below each image provide the amount of geometric variation from the mean head .
, the geometric variation has been added to the left half of the face , while the right side retains the mean head geometry .
the percentage given below refers to the geometric variation on the asymmetric side of the face .
asymmetric faces were created by fusing one side of a face with varying amounts of facial identity to the complimentary side of the mean ( i.e. , 0% identity ) face . to ensure that there were no artifacts due to contour misalignments between the two face halves , a weighted average of the gray levels 30 pixels from the vertical midline in each half was calculated to create a smooth contour .
this fusion was completed for six identity strengths ranging from 0 to 10% for each individual , and also for the left and right sides of the individual face .
an example of one asymmetric face trajectory is presented in figure 1b , where the asymmetry is added to the left half of the face .
the percentages provided below each exemplar correspond to the variation from the mean head radius in the asymmetric half of the face .
the rf patterns that were used as a nonface shape control stimulus were generated as described by wilkinson et al .
the shapes are generated by applying a sinusoidal modulation about a mean circle with a cross - sectional luminance profile of a fourth derivative gaussian ( figure 4a , top ) .
the frequency of the modulation determines the number of lobes in the shape , and the amplitude determines the strength of the shape and is expressed as the percent variation from the mean shape ( i.e. , circular ) radius . in the current study , we generated rf patterns at two phases ( 90 and 270 ) with a rf of 8 , which is well above the frequency for optimal performance ( wilkinson et al . , 1998 ) .
for each phase , we generated six shapes with shape strengths ranging from 0 to 1.5% mean shape radius .
to create asymmetrical rf patterns , we used the same technique that was used to create asymmetrical faces .
rf patterns were bisected along the vertical midline , and were fused to the complimentary side of the mean circle .
again , the contour between each side was smoothed by taking the weighted average of the middle 30 pixels .
an example of the asymmetric rf pattern is provided in the bottom of figure 4a .
two sets of experiments were conducted to determine whether the sensitivity to facial asymmetry was affected by stimulus manipulations that have been demonstrated to affect face identification .
first , asymmetry thresholds were evaluated with faces that were inverted 180 ( figure 5a ) .
all other aspects of the stimuli in this condition were identical to the upright faces used in the primary experiment .
second , asymmetry thresholds were evaluated for faces that were constructed about a nonmean face . in these stimuli , the amplitudes that define the identity of an individual face were added to a face with 12% identity variation from the mean .
increasing asymmetry was then added to the left or right side of the nonmean face in the same manner as it was in the faces constructed about the mean .
an example of an asymmetric face constructed about a nonmean face is provided in figure 6b .
individual thresholds for discriminating radial frequency ( rf ) patterns in both identity and asymmetry conditions .
subjects were required to either discriminate a full rf8 pattern from a mean circle ( top ( a ) : identity condition ) , or a pattern where the rf8 contour was only added to one half of the shape ( bottom ( a ) : asymmetry condition ) .
( b ) identity ( left ) and asymmetry ( right ) thresholds for rf patterns for five individual subjects .
two subjects ( lh and am ) had equivalent thresholds for each condition , whereas the other three subjects had lower thresholds for the identity condition than for the asymmetry condition .
( a ) thresholds for detecting asymmetry were compared when the faces were presented upright ( top ( a ) ) , or when rotated 180 ( bottom ( a ) ) .
( b ) individual asymmetry thresholds for three subjects for the upright ( left ) and inverted ( right ) faces .
asymmetry thresholds for faces constructed using either the mean or a nonmean face as a geometric base .
( a ) asymmetric faces were created by adding geometric asymmetry to either the mean face ( top ( a ) ) , or a nonmean face possessing 10% geometric identity ( bottom ( a ) ) .
the percentages given below refer to the amount asymmetry added to the face . in both examples ,
( b ) individual asymmetry thresholds for three subjects for the mean ( left ) and nonmean ( right ) base faces .
stimuli were presented on an intel mac mini with a 1.66 ghz core duo processor and a 17 inch seanix monitor with a resolution of 1024 1280 and a 75 hz refresh rate .
stimulus generation and presentation was accomplished using psychtoolbox functions ( brainard , 1997 ) and custom software developed in the matlab programming environment .
subjects viewed the stimuli from a distance of 57 cm , which was maintained by the use of a chin rest .
thresholds were determined using a two interval - forced choice procedure , where one interval contained either a face from the identity trajectory for one individual ( identity condition ) or a face from the asymmetric trajectory ( asymmetry condition ) , and the other interval contained the mean or symmetric face .
subjects responded , by keyboard , which interval contained the individual or asymmetric face . to ensure that discriminations were not based on local changes in contour features , the size of the faces across the two presentation intervals was randomly jittered from between 2.3 2.9 and 4.6 5.8. each face was presented for 120 ms , followed by a noise mask presented for 200 ms .
the noise mask was matched to the size of the preceding face , and was bandpass filtered to contain the same peak spatial frequency as the faces . within each experimental run
four individual identity trajectories were created , with identity strengths ranging from 0 to 15% mean head radius in 3% increments . in the asymmetry condition ,
two individual identities were used ; however , for each identity , there were two asymmetry ( i.e. , left and right ) conditions resulting in four asymmetry trajectories in each experimental run .
thresholds did not differ between the left and right asymmetry conditions , and data across these conditions were collapsed into simply the asymmetry
therefore , in each experimental run , there were 24 total face stimuli ( four trajectories six increments ) , which were each presented 20 times for a total of 480 trials .
percent correct data for the 6 identity strength increments ( x ) were fit with a quick ( 1974 ) function of the form :
( 2)f(x)=10.52(xa)
where corresponds to threshold ( 75% correct ) and reflects the slope of the function .
all of the data presented reflect the average threshold estimation for a minimum of three experimental runs for that condition , where each of the different runs contains faces taken from different identity trajectories .
although statistical significance was determined and presented using standard parametric statistics , significant effects were confirmed using non - parametric analyses ( not presented ) .
five observers participated in the experiments evaluating face and rf asymmetry thresholds , whereas three observers participated in the conditions evaluating asymmetry thresholds with inverted and nonmean faces .
the authors ( na and cg ) participated in all of the conditions . all other observers were nave to the purpose of the experiments .
identity and asymmetry discrimination thresholds were determined for five observers . for all five observers , thresholds for discriminating the identity of a face were higher than thresholds for discriminating asymmetric from symmetric faces .
bars on the left - hand side of the graph denote threshold values for accurately discriminating individual identities from the mean face . on average , subjects required 7.58 1.08% geometric change from the mean to reliably discriminate the individual face , which is similar to threshold values that have been observed with these synthetic faces previously ( e.g. , wilson et al . , 2002 ) .
bars on the right - hand side of the graph display the average thresholds for discriminating an asymmetric from a symmetric face . for all five subjects , thresholds for discriminating the asymmetric face are considerably lower than they are for discriminating face identity ; on average , subjects only required a 3.37 0.48% geometric variation to discriminate the asymmetric faces .
in fact , thresholds for every subject measured are 2 times lower for asymmetry discrimination than they are for identity discrimination .
a paired t - test confirmed that the thresholds for asymmetry discrimination are reliably lower than the thresholds for identity discrimination ( t(4 ) = 4.9422 , p < 0.01 ) .
these results demonstrate that considerably less geometric variation is required to reliably detect facial asymmetry .
individual thresholds for discriminating identity ( left ) and asymmetry ( right ) from a symmetric mean face .
for all five observers , thresholds for discriminating asymmetry were 2 times lower than thresholds for discriminating identity .
one potential feature that could be contributing to this benefit for detecting asymmetry could come from the nature of the information in the stimuli themselves .
it is possible that facial asymmetry is more salient because there is a greater overall difference in the amount of information between the left and right sides of the face . with this logic ,
the better performance observed with asymmetric faces may be due to a difference in the amount of information available for a visual decision , as opposed to an advantage for asymmetric faces per se . to determine if there is in fact a difference in the amount of information between the two face types , we analyzed all of the face images to determine how many pixels differed between the left- and the right - half of the face .
this method of analysis was straightforward : the face images were bisected and matched to one another in matlab ( figure 3a ) .
if the pixels in the two corresponding images were the same gray level , then a zero was assigned to that spatial location , and if the pixels were a different gray level , then a one was assigned .
this yielded a difference image that provides a quantitative measure of the difference between the two face sides : the greater the number of difference pixels , the greater the difference in overall information between the left and right sides of the face .
we analyzed all of the images used in the current study , at all identity increments , and plotted the overall pixel difference for all of the images , in both the identity and asymmetric faces .
the mean pixel difference for all identity increments for both face types is presented in figure 3b .
the important result here is that there is no significant difference in the number of difference pixels between the identity and asymmetry faces .
thus , the advantage for detecting asymmetry can not be explained by a difference in the overall amount of information between the two face types : on average , the amount of geometric difference between the two face halves is the same for both the identity and asymmetry faces .
( a ) all face stimuli were divided along the vertical meridian ( left ( a ) ) , and the left and right halves were compared to determine the number of pixels that differed in each face half ( right ( a ) ) .
this was performed for all faces from the identity sets ( top ( a ) ) , and the asymmetry sets ( bottom ( a ) ) . the total number of difference pixels was then summed over each face stimulus . (
b ) average pixel differences between the left and right halves as a function of identity strength for faces with varying geometry identity ( black circles ) and asymmetry ( gray circles ) .
there is no significant difference in the overall amount of information between the left and right halves of the face for both face types .
it is also possible that this advantage for discriminating asymmetry could reflect a more general object - processing advantage , as opposed to a face - specific benefit for asymmetry . to evaluate this possibility , we measured discrimination thresholds for rf patterns in five subjects .
rf patterns are an ideal control stimulus in the current set of experiments , because they are the basis shapes for the construction of the synthetic faces . in the current experiments , subjects discriminated rf patterns from a circle using a 2ifc paradigm identical to that used in the face experiments ( figure 4a ) .
subjects also discriminated the asymmetric rf pattern from a circle in the asymmetric condition , again using a paradigm identical to that used to measure facial asymmetry discrimination ( figure 4a ) .
thresholds in the rf task reflect the minimum rf amplitude required for subjects to discriminate the shape from a mean circle .
( na , cg and ap ) , thresholds for discriminating the full rf pattern from the mean circle are lower than they are for discriminating the asymmetric pattern . for the other two subjects ( lh and am ) , there is no difference in thresholds between the conditions .
overall , thresholds approached , but did not reach , statistical significance ( t(4 ) = 2.5275 , p = 0.065 ) .
regardless of the individual differences , none of the subjects were better at discriminating the asymmetric rf pattern relative to discriminating the full rf pattern .
this suggests that the asymmetry advantage does not extend to judgments of more basic shape patterns , and instead reflects a face - specific advantage for asymmetric faces .
given the finding that this advantage for asymmetry discrimination appears to be a face - specific effect , we also investigated whether or not it was disrupted by manipulations that have been demonstrated to disrupt face - specific processing .
the first manipulation that we investigated was the inversion effect , where it has been clearly demonstrated that simply inverting a face disrupts face processing ( yin , 1969 ) .
we also measured face asymmetry thresholds in inverted faces and compared those results to the asymmetry thresholds for upright faces .
thresholds were measured for three subjects ( na , cg and kw ) , where the experimental paradigm was identical to that used in the asymmetry task except for the fact that the faces were rotated by 180 ( figure 5a ) .
these results are presented in figure 5b , with the original upright face asymmetry thresholds also presented as a comparison .
the asymmetry thresholds for the inverted faces are not significantly different for the inverted faces ( t(2 ) = 0.32715 , p > 0.2 ) , suggesting that inversion does not affect facial asymmetry perception with these stimuli .
another feature of face processing that can affect identity thresholds is whether or not the identity of the face is geometrically close to the mean face .
specifically , some evidence suggests that sensitivity to face identity is greater when the geometric features of the face are closer to average than when they are geometrically distant from average ( wilson et al . , 2002 ; but see rhodes , maloney , turner , & ewing , 2007 ) .
we also investigated whether or not face asymmetry discrimination depended on the geometric distance of the face from the mean . to this end , we generated asymmetries in faces that possessed 12% variation from the mean , which is well above identity detection thresholds for these faces .
geometrically asymmetric information was then added to either the left or right side of the nonmean face ( figure 6a ) . in these stimuli
, a 0% geometric variation means that the face does not vary from the nonmean face that is used as a base , and the geometric variation refers to the amount of variation on the asymmetric side that is different from the nonmean base face .
thresholds for these nonmean asymmetric faces are presented in figure 6b for three subjects ( na , cg and kw ) .
once again , asymmetry thresholds for the nonmean faces were not statistically different from faces that were asymmetric about the mean face ( t(2 ) = 1.1099 , p > 0.2 ) .
facial asymmetry discrimination , therefore , does not depend on where the face is located in geometric face space .
our results demonstrate that human observers are more sensitive to changes in facial asymmetry than they are to changes in face identity when discriminating from a mean face .
this is somewhat surprising , given the fact that there is actually less geometric variation in the asymmetric faces , since the variation is only added to one side of the face .
this advantage for discriminating asymmetry over just a general deformation from the mean does not extend to more simple geometric forms , suggesting that the advantage for discriminating asymmetry is a face - specific effect .
however , the advantage for discriminating asymmetry is not influenced by manipulations that typically affect face - specific processes , namely face inversion and nonmean face trajectories .
taken together , these results suggest that discriminating face asymmetry is a behavior that does not rely on the same mechanisms that support other face - specific behaviors ( for example , face identification ) .
the advantage for judging asymmetry could arise because of differences in the stimuli themselves . distorting
the symmetry of the face could mean that there is more of a visual difference between the left and right sides of the face , which would make it visually easier to discriminate .
we evaluated this possibility by analyzing the overall pixel difference between the left and right sides of the face in both the identity and asymmetry stimulus sets .
we found that there was no overall difference in the amount of change between the left and right sides of the face for the two stimulus sets .
this form of stimulus evaluation is a global analysis : the difference pixels simply reflect the overall number of pixels that differ , but not how those differences are spatially distributed .
it is possible that while the overall difference between the left and right sides of the face is the same for both asymmetric and identity faces , the asymmetric faces possess greater differences between specific regions of the face ( i.e. , local differences ) .
these could then be used as cues for discriminating the asymmetric face from the mean face .
it is important to note here , however , that even if subjects are using local cues to discriminate asymmetric faces , these are cues that are not traditionally thought to be involved in face identification ( a global process ) .
if these potential local cues are then contributing to asymmetry discrimination , this still supports the idea that asymmetry relies on mechanisms that differ from those that are involved in traditional face identification .
we found that thresholds for discriminating an asymmetric rf pattern from circularity were higher than thresholds for discriminating a modulated shape from circularity .
these results demonstrate that asymmetry perception is not in and of itself an easier task than shape discrimination , but instead appears to demonstrate a face - specific advantage .
these results also suggest that superior performance in the asymmetry condition does not reflect the use of local midline cues for discrimination , as subjects would have exhibited similar advantages with asymmetric figures in the rf condition if midline cues were being used .
our finding that performance is better when the deformation extends across the entire stimulus is consistent with previous research demonstrating that discrimination thresholds for rf patterns improve as the number of deformation cycles increases ( loffler , wilson , & wilkinson , 2003 ) .
these results support the idea that the visual system pools contour information for shape discrimination , and that as more information can be pooled , individuals become more sensitive to shape information . in our study , thresholds for three subjects were 2 times better when the deformation extended around the entire shape , and for two subjects thresholds did not change between the two conditions . under no circumstance
did we see an improvement when the deformation was only added to half of the shape .
thus , the improvement with pooling that is observed with rf patterns does not appear to extend to processing contour information in face discrimination .
our finding that face asymmetry discrimination was unaffected by inversion is consistent with the findings of little and jones ( 2006 ) , who also found that symmetry detection was unaffected by face inversion .
( 2005 ) , who found subjects were less accurate when discriminating chimeras from their normal counterparts when the faces were inverted as opposed to upright .
two critical differences between our study and that of rhodes et al . may account for these results .
first , the stimuli used by rhodes et al . were symmetric faces that were generated using naturalistic photographs , and where the hairlines of both the normal and symmetric faces possessed symmetric hairlines . as a result ,
the only cues for asymmetry with these faces were in either the feature placement , and/or the chinline of the faces . in the faces used in the present study ,
judgment of asymmetry when a global asymmetry is applied to the principal components of curvature in a head form is better than when the asymmetry is applied to components that are not typically seen in head shapes ( wilson & wilkinson , 2002 ) .
perhaps the fine - tuned differences in asymmetry with the feature placement in the chimeras may be more susceptible to inversion , whereas the more global differences in asymmetry with our stimuli may overcome any effects of inversion . moreover , performance in the rhodes et al .
study evaluated the ability for subjects to discriminate chimeric faces from their normal counterparts , without varying the amount of symmetry . as we were able to measure the minimum amount of asymmetry required to discriminate an asymmetric face ( as opposed to classifying symmetric from asymmetric faces ) , it is possible that we are tapping into a different visual skill , one that is less susceptible to face inversion . the current study has investigated sensitivity to bilateral face symmetry , where the axis of symmetry is located through the midline of the face .
bilateral symmetry is a feature that is often implicated in judgments of attractiveness or overall health ( e.g. , grammer & thornhill , 1994 ; scheib et al , 1999 ) .
however , another way that facial asymmetry can arise is through slight changes in head orientation ; when viewing a face from the side , the left and right views of the face also become more asymmetric relative to one another .
the type of information that is conveyed through asymmetries that occur due to head orientation is different from what we have described here ; namely , asymmetric changes due to head rotations can provide important social cues for attention direction ( e.g. , langton , watt , & bruce , 2000 ) .
moreover , the type of asymmetry that arises through slight head rotation is different from bilateral asymmetry , in that slight head rotation can be modeled as an affine transform ; the resulting asymmetry can be rectified by the visual system as an object invariance .
the bilateral asymmetry that is introduced here , on the other hand , reflects a non - affine transformation .
as such , asymmetry due to head rotation reflects a different form of asymmetry from the type that we have investigated here . whether or not the advantage that we observed for discriminating asymmetry from identity would extend to asymmetries due to head orientation can not be answered from the current study .
however , given our exquisite sensitivity to social attention cues such as eye gaze ( e.g. , see nummenmaa & calder ( 2009 ) for a review ) , and the strong sensitivity that we observed in the current study for bilateral asymmetry , we would predict an advantage for discriminating asymmetries due to head orientation as well . we propose that the difference between judgments of facial asymmetry and facial identity suggests that both visual skills rely on functionally different neural mechanisms . while the role of the ffa in face processing has been well established , other evidence suggests that a network of visual regions is involved in the variety of skills that are required for making different judgments of faces .
for example , the distributed model of face processing proposes that different cortical regions are involved in processing invariant aspects of faces as opposed to variant aspects of faces ( haxby , hoffman , & gobbini , 2000 ; winston et al . ,
2004 ) and/or the building - blocks of face processing ( nichols , betts , & wilson , 2010 ) .
fmri evidence also suggests that the perception of face symmetry may rely upon activity in the right ofa ( chen , kao , & tyler , 2007 ) .
taken together , these results suggest that discriminating facial asymmetry relies on functionally different mechanisms , and our results suggest that these mechanisms may be more sensitive to geometric change than those involved in perception of facial identity . | bilateral symmetry is a facial feature that plays an important role in the aesthetic judgments of faces .
the extent to which symmetry contributes to the identification of faces is less clear .
we investigated the relationship between facial asymmetry and identity using synthetic face stimuli where the geometric identity of the face can be precisely controlled .
thresholds for all observers were 2 times lower for discriminating facial asymmetry than they were for discriminating facial identity .
the advantage for discriminating asymmetrical forms was not observed using nonface shape stimuli , suggesting this advantage is face - specific .
moreover , asymmetry thresholds were not affected when faces were either inverted or constructed about a nonmean face .
these results , taken together , suggest that facial asymmetry is a characteristic that we are exquisitely sensitive to , and that may not contribute to face identification .
this conclusion is consistent with neuroimaging evidence that suggests that face symmetry and face identity are processed by different neural mechanisms . |
atlantoaxial instability can be caused by various reasons like trauma , malignancy , congenital malformation , or inflammatory diseases and it is usually treated by reduction and stabilization of the c1-c2 joint . since the first description of the sublaminar wiring technique by gallie in 19399 ) , various modification of the techniques had been suggest by brooks , sonntag , magerl and harms3,6,11,17,23,31 ) .
although these techniques had been developed and modified to achieve the better result of stabilization of atlantoaxial complex , the operations are still challenging due to devastating complications or technical difficulties . based on individual screw placement in c1 and
c2 suggested by harms and melcher17 ) , we have added our new technique to reinforce stabilization and accomplish successful bone fusion . with an expectation to acquire better outcome , less complication , and technical facility ,
we have performed this technique to thirteen patients over the past 4 years . in this study , we present our technique for posterior c1-c2 fusion by using miniplate for interlaminar fusion which supplements c1 lateral mass and c2 pedicle screw fixation . in addition , a retrospective review of thirteen patients treated by this technique is also presented .
thirteen patients underwent the procedure from march 2006 to october 2010 ( table 1 ) .
seven of these patients had odontoid process fracture with transverse ligament injury , four had rheumatoid arthritis and one had os odontoideum .
eight patients were approached with paramedian incision for the procedure and five patients including c1 - 2 vertical dislocation injury was approached with classical midline incision .
after the operation , all patients underwent computed tomography or plain x - ray with dynamic image during the follow - up except one with odontoid process fracture .
three criteria were evaluated with these images : 1 ) presence of bridging bone in computed tomography ; 2 ) absence of lucency around implant ; 3 ) less than 2 degrees of segmental motion and less than 2 mm change of interspinous distance of atlantoaxial spine on its dynamic view . under general anesthesia ,
the patient was placed in the prone position and the neck was held in alignment with mayfield .
bilateral paramedian incision was made 1.5 - 2 cm off to midline and muscle was dissected with the use of metrx quadrant system ( medtronics ) ( fig .
the c1-c2 joint was exposed and opened by dissection over the superior surface of the c2 pars interarticularis .
the dorsal root ganglion of c2 was retracted in a caudal direction to expose the entry point for the c1 screw , which was in the middle of the junction of the c1 posterior arch and the midpoint of the posterior inferior part of the c1 lateral mass .
then the pilot hole was drilled in a slightly convergent trajectory in an anterior - posterior direction and parallel to the plane of the c1 posterior arch in the sagittal direction , with the tip of the drill directed toward the anterior arch of c1 .
the drilling was accomplished with guidance from intraoperative landmarks and the lateral view of fluoroscopy .
the hole was tapped and a 4 mm vertex polyaxial screw ( medtronics ) of an appropriate length was inserted bicortically into the lateral mass of c1 . a number 4 penfield was used to delineate the medial border of the c2 pars interarticularis , and the entry point for placement of a c2 pedicle screw was marked with a high speed burr .
the entry point of c2 pedicle screw was in the cranial and medial quadrant of the isthmus surface of c2 .
the pilot hole was prepared with a 2 mm drill bit , just perforating the opposite cortex .
the direction of the bit was approximately 20 to 30 in a convergent and cephalad direction , guided directly by the superior and medial surface of the c2 pedicle .
the hole was tapped , and a 4 mm polyaxial screw of the appropriate length was inserted bicortically .
if necessary , reduction of the c1 ring was performed by manipulating c1 and c2 using the screws , followed by fixation to the rods to maintain the alignment .
then the posterior arch of c1 and c2 lamina were decorticated and tricortical bone taken from the posterior iliac crest was placed between decorticated surfaces of c1 and c2 .
5 to 7 mm length screws were placed on inferior surface of c1 posterior arch , center of tricortical bone and c2 lamina bilaterally ( fig .
2 ) . intraarticular fusion could be also performed by decorticating the joint surfaces under direct vision .
patients were mobilized on the first postoperative day and wear a soft cervical collar for 2 to 3 weeks .
thirteen patients underwent the procedure from march 2006 to october 2010 ( table 1 ) .
seven of these patients had odontoid process fracture with transverse ligament injury , four had rheumatoid arthritis and one had os odontoideum .
eight patients were approached with paramedian incision for the procedure and five patients including c1 - 2 vertical dislocation injury was approached with classical midline incision .
after the operation , all patients underwent computed tomography or plain x - ray with dynamic image during the follow - up except one with odontoid process fracture .
three criteria were evaluated with these images : 1 ) presence of bridging bone in computed tomography ; 2 ) absence of lucency around implant ; 3 ) less than 2 degrees of segmental motion and less than 2 mm change of interspinous distance of atlantoaxial spine on its dynamic view .
under general anesthesia , the patient was placed in the prone position and the neck was held in alignment with mayfield .
bilateral paramedian incision was made 1.5 - 2 cm off to midline and muscle was dissected with the use of metrx quadrant system ( medtronics ) ( fig .
the c1-c2 joint was exposed and opened by dissection over the superior surface of the c2 pars interarticularis .
the dorsal root ganglion of c2 was retracted in a caudal direction to expose the entry point for the c1 screw , which was in the middle of the junction of the c1 posterior arch and the midpoint of the posterior inferior part of the c1 lateral mass .
then the pilot hole was drilled in a slightly convergent trajectory in an anterior - posterior direction and parallel to the plane of the c1 posterior arch in the sagittal direction , with the tip of the drill directed toward the anterior arch of c1 .
the drilling was accomplished with guidance from intraoperative landmarks and the lateral view of fluoroscopy .
the hole was tapped and a 4 mm vertex polyaxial screw ( medtronics ) of an appropriate length was inserted bicortically into the lateral mass of c1 . a number 4 penfield was used to delineate the medial border of the c2 pars interarticularis , and the entry point for placement of a c2 pedicle screw was marked with a high speed burr .
the entry point of c2 pedicle screw was in the cranial and medial quadrant of the isthmus surface of c2 .
the pilot hole was prepared with a 2 mm drill bit , just perforating the opposite cortex .
the direction of the bit was approximately 20 to 30 in a convergent and cephalad direction , guided directly by the superior and medial surface of the c2 pedicle .
the hole was tapped , and a 4 mm polyaxial screw of the appropriate length was inserted bicortically .
if necessary , reduction of the c1 ring was performed by manipulating c1 and c2 using the screws , followed by fixation to the rods to maintain the alignment .
then the posterior arch of c1 and c2 lamina were decorticated and tricortical bone taken from the posterior iliac crest was placed between decorticated surfaces of c1 and c2 .
5 to 7 mm length screws were placed on inferior surface of c1 posterior arch , center of tricortical bone and c2 lamina bilaterally ( fig .
2 ) . intraarticular fusion could be also performed by decorticating the joint surfaces under direct vision .
patients were mobilized on the first postoperative day and wear a soft cervical collar for 2 to 3 weeks .
there was no case of neurologic deterioration after surgery or at follow - up related to the procedure .
therefore , fusion rate of this technique was 91.7% and considered as 100% , clinically .
a 40-year - old female was transferred from rheumatology department due to the posterior neck pain for 2 years .
3 ) . after taking ct and mri she was diagnosed to c1-c2 instability due to rheumatoid arthritis .
bilateral paramedian incision was made 1.5 - 2 cm off to midline and muscle was dissected with the use of metrx quadrant system .
the bleeding from epidural venous plexus impeded the dissection but it was controlled with a combination of gelfoam powder and thrombin . after the screw fixation on c1 and c2 , autologous bone harvested from her iliac bone was placed between c1-c2 lamina and fixed with miniplate . after 4.5 months we could confirm bone fusion through ct scan ( fig .
a 54-year - old female was admitted via emergency room with posterior neck pain after traffic accident .
preoperative x - ray after prone positioning revealed normal alignment of atlantoaxial segment so intraoperative reduction of c1-c2 was unnecessary .
after the screw fixation on c1 and c2 , autologous bone harvested from her iliac bone was placed between c1-c2 lamina and fixed with miniplate .
after medical treatment with analgesics , her condition was improved and she was able to be discharged with soft neck collar .
a 40-year - old female was transferred from rheumatology department due to the posterior neck pain for 2 years .
3 ) . after taking ct and mri she was diagnosed to c1-c2 instability due to rheumatoid arthritis .
bilateral paramedian incision was made 1.5 - 2 cm off to midline and muscle was dissected with the use of metrx quadrant system .
the bleeding from epidural venous plexus impeded the dissection but it was controlled with a combination of gelfoam powder and thrombin . after the screw fixation on c1 and c2 , autologous bone harvested from her iliac bone was placed between c1-c2 lamina and fixed with miniplate . after 4.5 months we could confirm bone fusion through ct scan ( fig .
a 54-year - old female was admitted via emergency room with posterior neck pain after traffic accident .
preoperative x - ray after prone positioning revealed normal alignment of atlantoaxial segment so intraoperative reduction of c1-c2 was unnecessary .
after the screw fixation on c1 and c2 , autologous bone harvested from her iliac bone was placed between c1-c2 lamina and fixed with miniplate .
after medical treatment with analgesics , her condition was improved and she was able to be discharged with soft neck collar .
traditionally , the atlantoaxial instability had been treated using c1 - 2 posterior wiring and bone graft .
because of the high mobility of the c1 - 2 motion segment , fusion rates at this level have been substantially lower than those at the subaxial spine . therefore , since the first description of the sublaminar wiring technique by gallie in 19399 ) , various modification of the techniques like wedge compression method and interspinous wiring method had been suggest by brooks and sonntag3,6,31 ) .
although the posterior wiring procedure is easy to accomplish , this technique carries possible risk of dural or neuronal injury during procedure and the halo vest immobilization is required to accomplish successful bone fusion . since the transarticular screw fixation had suggested by magerl and seemann in 198723 ) , supplemental halo brace became no longer necessary due to immediate rigid multidirectional stability25 ) .
although the transarticular screw fixation provided superior stability and bone fusion rate to wiring or the use of halifax clamps13,14,16,19,25,27,33 ) , this technique requires meticulous preoperative planning and precise operative techniques are mandatory to achieve successful result and to avoid vertebral artery injury . in 2001 , harms and melcher17 ) described a technique for individual fixation using polyaxial screw and rod system and similar study was published by goel et al . in 200211 ) . by using c1 lateral mass and c2 pedicle screw with polyaxial screw followed by fixation to the rods to maintain its alignment , intraoperative reduction of the c1 ring became possible if necessary and
moreover , this procedure has shown to confer excellent stability in biomechanical studies18,21,22 ) , so the result of the procedure was good as transarticular screw fixation , especially during flexion and extension21,22 ) .
the fusion rates of brooks and gallie technique are 70% to 85%1,7,29 ) although the rate ranges from 60% to 100% in smaller studies1,2,12,26 ) .
the fusion rate among the patients with fracture is higher with interspinous method suggested by sonntag6 ) . with transarticular screw fixation by magerl ,
c1 lateral mass and c2 pedicle screw fixation with polyaxial screw and rod system have been considered to show similar outcome with transarticular screw fixation11,17 ) , however , to maximize the bone fusion rate of these technique , the combination with either a gallie or a brook 's fusion is necessary28 ) .
possible complications of sublaminar wiring technique is neuronal injury during the wire passage , especially at the c2 level where the spinal canal narrows compared to c1 level4 ) .
wires and instrumentation provide only temporary fixation , therefore halo vest immobilization is necessary for successful osseous fusion6 ) . in spite of great advantage over the wiring technique ,
since this procedure requires preliminary reduction of c1 on c2 before the screw insertion and patient 's neck flexed to obtain proper guidance of the drill , in some cases , such as irreducible c1 on c2 or the patient with pronounced thoracic kyphosis , the feasibility is restricted .
in addition , sufficient space must be available in the pedicle for placement of the screw around the vertebral artery20,24,30 ) . a screw trajectory that is too lateral or too medial
the incidence of vertebral artery injury has been reported to range between 2.6% and 4.1%10,34 ) .
harm 's technique has technical advantage over transarticular screw fixation in the point of possible vertebral artery injury or the necessity of less operation field during the procedure .
however , the procedure has a same risk of neuronal injury with gallie method when they are combined with sublaminar wiring technique to achieve maximal bone fusion as previously described . in this study , we have performed harm 's technique with interlaminar fusion using autologous bone and miniplate to maximize bone fusion and minimize complications
. the mechanical strength of interlaminar wiring or interspinous wiring might be much superior to miniplate fixation .
however , in harm 's technique , wire does not need to support or stabilize c1-c2 segment itself , unlikely in gallie , brook , or sonntag 's technique .
because the c1-c2 segment is firmly stabilized with c1 lateral mass and c2 pedicle screw , the miniplate only needs to provide strength enough to stabilize autograft bone between the interlaminar space until successful bone fusion is accomplished .
the result of this study that bone fusion between interlaminar space were occurred in eleven out of twelve cases has proven the efficacy of miniplate as a stabilizing device of autograft bone between interlaminar space . even in case with failed bridging bone formation , the placement of miniplate was not changed .
we suspected less decorticated c1 posterior arch or soft tissue might hinder bridging bone formation . by using this miniplate fixation for interlaminar fusion , neuronal or dural injury
there is no need of dissecting cranial end of posterior arch of the atlas for lack of the necessity of wire passage on it .
dissection on c1 can be reduced to minimal because the exposure to only postero - inferior margin is required for miniplate fixation .
moreover , this procedure can be applied even when the posterior arch of atlas is compromised as long as the posterior arch is partially remained .
major complications of cervical surgery with posterior approach are due to dissection of midline structures .
we could assure that paramedian approach was possible with this technique because miniplate fixation was technically much easier than the wiring technique .
therefore , we could save nuchal structures which lead to reduction of possible complication after midline incision , result from extensive dissection of midline musculature32 ) .
yet this study has limitations because the size of data was small and the clinical outcome was not evaluated . in the future , the study with larger group and comparative study with other method will be needed and the assessment of clinical outcomes of patients should be evaluated .
posterior atlantoaxial fusion technique has been constantly developed and modified to achieve better fusion rate and to avoid complications . with our technique using miniplate to supplement c1 lateral mass and c2 pedicle screw fixation
, the procedure can be easier and safer to be performed due to the low rate of complications as well as technical facility .
moreover , as minimal invasive surgery becomes possible with this technique , better clinical outcome is expected . | objectiveto investigate the feasibility of c1 lateral mass screw and c2 pedicle screw with polyaxial screw and rod system supplemented with miniplate for interlaminar fusion to treat various atlantoaxial instabilities.methodsafter posterior atlantoaxial fixation with lateral mass screw in the atlas and pedicle screw in the axis , we used 2 miniplates to fixate interlaminar iliac bone graft instead of sublaminar wiring .
we performed this procedure in thirteen patients who had atlantoaxial instabilities and retrospectively evaluated the bone fusion rate and complications.resultsby using this method , we have achieved excellent bone fusion comparing with the result of other methods without any complications related to this procedure.conclusionc1 lateral mass screw and c2 pedicle screw with polyaxial screw and rod system supplemented with miniplate for interlaminar fusion may be an efficient alternative method to treat various atlantoaxial instabilities . |
tumor necrosis factor receptor - associated factors ( trafs ) act as adapter molecules controlling signaling pathways , such as nuclear factor kappa b ( nf-b ) , interleukin-1 receptor ( il-1r ) , toll - like receptor ( tlr ) , and transforming growth factor- ( tgf- ) [ 1 , 2 ] . for a long time
it is important that traf6 contributes to the cd40-mediated activation of nf-b and c - jun kinase ( jnk ) .
association of traf6 with cd40 is essential for cd40-mediated il-6 expression , which could explain the requirement for membrane - bound cd40 ligand to induce il-6 production by immunocytes . the survival , regulation , and activation of immunocyte and epithelial cell , signaling through cell surface receptors to activate nf-b and mitogen - activated protein kinases ( mapks ) through traf6 , are critical regulations of immune response .
unlike traf6 , the molecular mechanism of traf4 in multiple signaling pathways triggered by tnfr - related proteins remains enigmatic .
it has been indicated that traf4 augments nf-b activation through glucocorticoid - induced tnfr ( gitr ) expression on t cells , b cells , and macrophages . as a unique traf family member mediating signal transduction by tnf , il-1r , or tlr , it is found that traf4 acts as a positive effector of bone morphogenetic protein ( bmp ) and the tgf- signaling pathway .
the intestinal epithelium and immune cells in the gut establish active sites of immune reactivity .
breakdown of homeostasis between intestinal microbiota and the mucosal immune system , together with both environmental and genetic factors , leads to inflammatory bowel disease ( ibd ) .
nf-b and tlr are considered as nodal points in the suppression and/or recruitment of immune responses in ibd .
interestingly , although mechanisms of trafs in ibd are not yet fully studied , traf - related inflammatory mediators as tgf- or cd40 play critical roles in a wide array of cellular functions in ibd [ 9 , 10 ] . recent studies in immune cells and transgenic mice regarding the role of traf4 and traf6 have revealed that they share the same binding sites , yet comprehensive study of traf4 and traf6 in ibd is still lacking . based on the hypothesis that traf4 and traf6 may be activated prior to the clinical or endoscopic activation in patients with ibd , we sought to measure traf4 and traf6 expressions to explore their potential roles in ibd patients .
patients with pregnancy , colorectal resection for uc , disease involving only small bowel , poor bowel preparation as visible area of intestinal mucosa < 90% , or use of steroids , immunosuppressants , or infliximab before colonoscopic sampling were excluded . no diagnosis altered after at least 3 months of followup .
endoscopic score was evaluated using simplified endoscopic score in crohn 's disease ( ses - cd ) or baron score for patients with cd or uc , respectively .
human intestinal biopsies and blood samples were collected at division of gastroenterology and hepatology , shanghai jiao - tong university school of renji hospital medicine , in accordance , with guidelines of the research ethics committee of renji hospital , shanghai jiao tong university , school of medicine .
human tissue specimens were taken from both macroscopically inflamed and non - inflamed regions of the colon .
tissue specimens were put into liquid nitrogen within 10 minutes after biopsy for protein extraction or kept in rnalater ( qiagen ) for rna isolation .
human peripheral blood was separated into plasma and peripheral blood mononuclear cells ( pbmcs ) .
plasma was obtained using commercially edta - treated tubes ( gongdong medical technology co. , ltd . ) and pbmcs were isolated according to lymphoprep ( axis - shield poc as , norway ) protocol .
briefly , diluted blood was overlayed over 3 ml lymphoprep and centrifuged at 800 g for 20 mins .
then , pbmcs were kept in rnalater ( qiagen ) for rna isolation according to manufacturer 's protocol .
plasma was obtained following centrifugation of whole blood for 15 minutes at 2,000 g .
samples were analyzed using kits against traf4 and traf6 , according to the manufacturer 's specifications ( lanji biochemical and diagnostics , shanghai , china ) and a microtiter plate reader was used to read absorbance at 450 nm .
total rna in pbmcs and tissue samples were isolated with trizol reagent ( ambion ) according to the manufacturer 's protocol for cells and tissue .
the quantity and quality of rna were detected using a nanodrop 1000 ( nanodrop technologies , wilmington , de , usa ) .
primers were designed with primer 5.0 ( abi ) software and consequently synthesized by sango biotech ( shanghai ) co. , ltd .
the primer set for traf4 was 5-aggagttcgtctttgacaccatc-3 ( forward ) and 5-ctttgaatgggcagagcacc-3 ( reverse ) , with a product of 162 bps .
the primer set for traf6 was 5-cctttggcaaatgtcatctgtg-3 ( forward ) and 5-ctctgcatcttttcatggcaac-3 ( reverse ) , with a product of 140 bps .
the primer set of gapdh was 5-gtgaaggtcggagtcaacgg-3 ( forward ) and 5-cctggaagatggtgatgggat-3 ( reverse ) , which provided a product of 226 bps .
cdnas were produced with primescripttm rt reagent kit ( takara biotechnology dalian co. , ltd . ) .
briefly , reverse transcripts were incubated at 37c for 15 minutes and 85c for 5 seconds .
sybr premix ex taq kit was purchased from takara and real - time pcr reactions were done using a stepone plus device ( applied biosystems ) at 95c for 10 seconds followed by 40 cycles of 95c for 5 seconds and 60c for 20 seconds according to instruction of the sybr premix ex taq kit .
the expression levels of the target genes were normalized to gapdh with 2-ct method . for western blot analysis , pmbcs and tissue samples
were lysed in ripa buffer ( sigma ) containing protease inhibitors ( roche ) and agitated on ice for 30 minutes .
protein concentrations were determined using pierce bca protein assay kit ( pierce , wohlen , switzerland ) .
protein electrophoresis was performed according to the protocol of mini - protea iii ( bio - rad ) .
briefly , proteins were separated in 10% polyacrylamide gels ( tris / glycine ) and transferred onto polyvinylidene fluoride membrane ( millipore ) .
western blots were probed with antibodies against traf4 ( rabbit polyclonal anti - traf4 ; 1 : 1000 , santa cruz ) , traf6 ( mouse monoclonal anti - traf6 , 1 : 1000 , santa cruz ) , and -actin ( mouse monoclonal anti--actin ; 1 : 2500 , santa cruz ) .
statistical significance was determined using graphpad prism 5.0 for windows ( graphpad software , san diego , ca , usa ) .
a p < 0.05 was considered significant with either anova analysis or tukey 's multicomparison .
a two - sided fisher 's exact test or test was performed to analyze discrete variables .
from february 2007 to february 2010 , 40 cd patients , 42 uc patients , and 40 healthy controls were included in our present study .
patients with ibd indicated significantly lower body mass index ( bmi ) than healthy controls ( p < 0.0001 ) .
the uc group contained significantly more smokers than cd patients ( 22 : 8 , p = 0.0023 ) .
three patients with ileitis , 15 patients with ileocolitis , and 22 patients with colitis were enrolled in cd group .
thirteen patients with proctosigmoiditis , 20 patients with left sided colitis , and 9 patients with pancolitis were enrolled in uc group .
characteristics of included subjects were described in table 1 . to investigate the diagnostic value of traf4 and traf6 in ibd , we detected levels of soluble traf4 and traf6 in plasma of ibd patients .
we found that traf4 and traf6 were significantly higher both in patients with cd and uc than in healthy controls ( figures 1(a ) and 1(b ) ) .
however , only overexpression of soluble traf4 showed a significantly positive correlation with endoscopic disease activity index ( baron score ) in uc patients ( spearman 's r = 0.458 , p = 0.002 ) .
furthermore , we observed that traf4 showed a significantly diagnostic value in differentiating active ibd patients from healthy controls ( p < 0.0001 , figures 2(a ) and 2(b ) ) .
although traf6 also showed a significantly diagnostic value in differentiating active cd , uc from healthy controls , the lower area under the curve ( auc ) predicted a less diagnostic value than traf4 ( figures 2(c ) and 2(d ) ) . to identify gene expressions of traf4 and traf6 in pbmcs in patients with cd and uc , we isolated rna from pbmcs .
similar to their expression in plasma , traf4 and traf6 showed significantly higher levels both in patients with cd and uc than in healthy controls ( figures 1(c ) and 1(d ) ) ( all p < 0.0001 ) . given that segmental changes can exhibit inflammation in the colon in ibd patients and that intestinal segments in endoscopic remission can appear as histologic colitis , we determined the expressions of traf4 and traf6 both in inflamed and non - inflamed intestinal mucosae . unfortunately , 3 patients with only ileitis and nine patients with pancolitis were excluded based on exclusion criteria .
quantitative real - time pcr ( qrt - pcr ) was used to determine the gene expressions of traf4 and traf6 in inflamed and non - inflamed intestinal mucosae of ibd .
it was found that traf4 and traf6 expressions were significantly higher in inflamed intestinal mucosa of patients compared to normal mucosa of healthy controls ( all p < 0.0001 ) ( figures 3(a ) and 3(b ) ) .
interestingly , traf6 expressions were also significantly higher in non - inflamed tissue of ibd patients than in healthy controls , which may indicate potential preactivation of traf6 in ibd ( figure 3(b ) ) .
western blotting was used to measure protein expressions of traf4 and traf6 in inflamed and non - inflamed intestinal mucosae of ibd ( figure 4 ) .
our data indicate that only traf6 expressions were significantly higher in non - inflamed tissue of ibd patients than in healthy controls , although traf4 and traf6 protein expressions were significantly higher in inflamed intestinal mucosa of patients than in normal mucosa of healthy controls ( all p < 0.0001 ) ( figures 3(c ) and 3(d ) ) .
similar to their gene expressions , traf4 and traf6 protein expressions were significantly increased in inflamed intestinal mucosa compare to the non - inflamed mucosa or healthy controls .
also , traf6 protein expression was significantly higher in inflamed intestinal mucosa of ibd patients compared to healthy controls .
in the current study , we demonstrated that two members of the traf family , traf4 and traf6 , were activated in patients with ibd .
although both traf4 and traf6 showed potentially diagnostic value in differentiating active cd and uc from healthy controls , only traf6 could be pre - activated in non - inflamed tissue of ibd patients .
although trafs have similar overall structural features including a c - terminal receptor - binding domain and a leucine - zipper domain , their own structural difference leads to distinctive interaction with receptors [ 13 , 14 ] .
growing evidence indicates that trafs are regulated not only by their own structural features but also by the nature of their interactions , recruitment , or localization .
early overexpression studies clearly indicated that traf6 contributes to the cd40-mediated activation of nf-b and other signaling molecules .
nf-b activity has been upregulated in lamina propria immune cells and in epithelia of the inflamed gut in ibd .
the nf-b pathway regulates inflammation , regulatory t - cell production , and dc function .
however , activation of important transcriptional regulators including nf-b and the stress - activated protein kinases ( sapks ) mediated by traf6 requires binding to cd40 .
the cytoplasmic cd40 binding domain for traf6 is necessary for the cd40-mediated activation of il-6 production in monocytes and macrophages , and the proinflammatory il6 biologic network is upregulated in active ibd .
moreover , experiments with immune cells or fibroblasts isolated from traf6-deficient mice indicate that traf6 is required for the cd40-mediated activation of not only nf-b but also jnk and p38 mapk signals [ 19 , 20 ] .
although cd40-mediated jnk activation in b cells seems to require cytoplasmic traf6 , traf6 mutants defective in cd40 binding were able to activate the jnk pathway and upregulate cd80 , indicating that traf6 may be able to contribute to certain jnk signals without the binding of cd40 .
jnk and mapk signaling pathways are involved in governing lymphocyte influx into the gut in ibd patients by regulating lymphocyte adhesion and transmigration .
furthermore , only one study screened potential alterations of the traf6 gene in a large number of cd and uc patients but failed to identify apparent disease - causing mutations .
their data suggested that traf6 may have essential roles in human biology that it might not tolerate any significant structural alterations .
dendritic cells from traf4-deficient mice exhibited reduced migration both in vitro and in vivo experiments .
this result indicates that traf4 could participate in immune functions by facilitating immune cell migration .
interestingly , traf4 increases nf-b activation through the gitr via a traf - binding site located in the cytoplasmic domain of gitr .
this domain is responsible for the inhibition of treg cells and the promotion of t - cell activation .
although no nf-b activity has ever been detected via traf4 overexpression , traf4 has been implicated as an upstream molecule that regulates the jnk pathway via interaction and activation of misshapen ( msn ) , a member of the sps1 protein kinase family . moreover
, traf4 positively regulates transforming growth factor ( tgf)- via potentiating bone morphogenetic protein ( bmp ) and nodal signaling .
accordingly , traf4 is upregulated in b cells following cd40 signaling , which suggests that traf4 affected downstream of cd40 pathway .
this may explain our result that traf4 expressions were not significantly different in non - inflamed tissue of ibd patients compared to healthy controls .
our understanding of traf4 and traf6 functions in the present study indicates that overlapping and unique functions will ultimately be attributed to each of the trafs . to our knowledge
, this paper may be the first one to combine analyzing traf4 and traf6 in ibd patients .
first , the present study did not include histological criteria to distinguish non - inflamed tissue from inflamed tissue , which may confuse the microscopically inflamed tissues with non - inflamed tissues .
second , the present study could not include laboratory data as esr or crp to assess activity of disease , which may lead to the potential bias of overreliance on clinical manifestations and endoscopic assessment .
collectively , our data showed similar and different expression patterns of traf4 and traf6 in patients with ibd . to date | in recent years , interests combining the exploration of tumor necrosis factor receptor - associated factor 4 ( traf4 ) and traf6 in immune cells and transgenic mice are emerging .
although it has been found that traf4 and traf6 share the same traf binding sites , comprehensive study of traf4 and traf6 in inflammatory bowel disease ( ibd ) is still lacking .
this paper shows similar and different expression patterns of traf4 and traf6 in patients with ibd .
the results indicate that traf4 and traf6 are overexpressed in ibd .
traf4 and traf6 play different roles in the pathogenesis of ibd .
moreover , traf4 may be an indicator of endoscopic disease activity of uc and traf6 preactivation can be detected in noninflamed colonic segments . |
female genital mutilation or clitoris cutting ( fgm ) is defined as the partial or total removal of clitoris and labia .
well known since antiquity in egypt , this practice is widespread in the world but mainly in africa [ 17 ] .
many factors related to tradition , sexual behavior in the males , and religious beliefs impact on fgm [ 811 ] .
the clinical observation of three cases : first in female newborn twins aged three weeks and second in an 8-year girl led us to carry out a prospective study on fgm in infants and young girls .
we studied the prevalence and etiologic factors in the pediatric ward at the general hospital of abobo , a suburb of abidjan .
our study also aimed to influence the parents of girls and the traditional circumcisers and practitioners to abandon the practice . not only are there laws prohibiting fgm , but there are later gynecological and obstetrical consequences of fgm .
our objectives were ( 1 ) to identify fgm in infants and young girls seen in our clinic , ( 2 ) to describe the sociocultural context of young girls who had undergone fgm , ( 3 ) to assess the mothers ' fgm status and attitudes regarding the practice , ( 4 ) and to determine the clinical issues in terms of immediate or later complications .
the general hospital of abobo is the premier public health entity that takes care of many patients of abobo and others from periurban areas of abidjan .
this includes about 1,500,000 inhabitants or 20% of the population of the city of abidjan .
eligible for the survey were infants and young girls seen at the hospital for any reason and whose mothers agreed verbally or by written consent to enter the survey and answer the questionnaire items . from 16 april to 16 december , 2007 , during eight months , 409 infants and young girls aged from 1 to 14 years and their mothers entered the prospective study .
these examinations were complemented by a questionnaire comprising four groups of items : ( 1 ) patient 's identification with age , native region ( north , south , east , west , and center of the country ) , ethnic group , religion , and nationality ; ( 2 ) history and circumstances of fgm practice , age at fgm procedure , observance of ritual ceremony or not , the individuals behind the decision of fgm practice , and the motivations ; the circumciser ( childbirth attendant or matron ) , tools used for fgm , and the occurrence of immediate complications such as bleeding , the medicines used to heal the wounds , the rituals observed during the fgm ceremony , time elapsing before full wound healing , and the fgm status of the mothers themselves ; ( 3 ) evidence of fgm by a physical general examination including the genitalia and classification of fgm , when present , according to the world health organization 's ( who ) classification : these are type i : sunna partial or total removal of the clitoris and/or the prepuce or excision , type ii : partial or total removal of the clitoris and the labia minora , with or without excision of the labia majora ( clitoredectomy ) , type iii : narrowing of the vaginal orifice with creation of a covering seal by cutting and apposing the labia minora and/or the labia majora with or without excision of the clitoris ( infibulations or pharaonic circumcision ) , and type iv : all other harmful procedures to the female genitalia for nonmedical purposes piercing , pricking , incising , scraping , and cauterization of the genitalia area ( unclassifiable ) .
ethics of our study . the general hospital consultative committee that evaluates the relevance , feasibility , confidentiality of the information obtained , and ethnical aspects of clinical research reviewed our protocol of research and gave its permission .
once the mother 's oral or written consent was obtained , the same female physician organized the questionnaire , performed the physical examinations , and filled out the data sheets during both inpatients and outpatients consultations .
sixty of 409 infants and young girls ( 15% ) were diagnosed as having undergone fgm .
their age distribution at the time of consultation or hospitalization was between 1 and 5 years : 19 , ( 32% ) between 5 and 10 years : 29 ( 48% ) and between 10 and 15 years : 12 ( 20% ) .
the baseline characteristics on epidemiological aspects were the earlier age at fgm practice , 19 infants under one - year old ; women were the individuals behind the decision of fgm practice ( 96.60% ) ; several west african ethnic groups from cote d'ivoire , mali , burkina faso , benin , and niger were implicated ; muslim families 59/60 and the illiterate or low educational level of the parents 81% and 87% , respectively in mothers and fathers were found as major factors .
the practitioners were traditional circumcisers ; no nurses , midwives , or physicians were involved .
pain , fever , and minimal bleeding were the main symptoms and signs disclosed by the mothers surveyed .
there was a potential relative risk of undergoing type ii mutilation for those under five years of age . amongst the mothers , 250 women out of 409
had had fgm ( 61.1% ) . among them , 151 had their daughters ( 60.4% ) undergone the procedure .
the details of sociocultural characteristics of our samples and the clinical findings of our patients are reported on the tables 1 , 2 , and 3 and figure 1 shows a fgm type ii in a 1-year - old peulh female .
the main associated factors were as follows : women were the decision makers relative to fgm ; in 97% of cases , it was a grandmother , mother , or aunt who initiated the operation .
fgm was encountered most often in the communities of three countries with a relative high rate in some ethnic groups as the malinke and senoufo .
most families were muslim ( 98.3% ) and most parents were illiterate , 81% and 87% in mothers and fathers , respectively .
previous reports on fgm in west african women [ 1317 ] gave rates varying from 45 to 60% in the general population and 20 up to 87% in northern and western regions of the country .
rates were high among the dan , malinke , w , and senoufo ethnic groups .
the proportion of young girls has been estimated by oula in his report to be about 500 females .
those were 31% for 155 children between 0 and 5 years ; 31.4% for 157 between 12 and 16 years of age ; and 38.6% in 193 women .
the religions were distributed in this way : christians : 55.91% , muslims : 43.34% , and animists 0.75% .
the decision makers were mainly women : grandmothers ( 71.6% ) , mothers ( 25.0% ) , and fathers ( 3.4% ) . in a survey carried out in 38,816 egyptian young school girls
the motivations were religion : 33.4% , cleanliness for girls : 18.9% , cultural and ancient tradition : 17.9% , and chastity 15% .
compared to the study of snow et al . in nigeria , similar characteristics had been found amongst young girls and women between 15 and 49 years victims of fgm and interviewed : age at fgm prior one year 371 ( 68.3% ) , between one and ten years 43 ( 7.9% ) , and ten to twenty years 88 ( 16.3% ) .
the religious context in this study was pentecostals : 562 ( 33.1% ) , protestants 277 ( 16.3% ) , catholics 613 ( 36.1% ) , muslim 100 ( 5.9% ) , and others 146 ( 8.6% ) .
on the other hand educational level of the surveyed girls was distributed as follows : primary : 330 ( 19.3% ) , secondary : 533 ( 32.2% ) , tertiary 767 ( 44.9% ) , and none 77 ( 4.5% ) showing the inhomogeneous and spatial distribution of sociocultural factors in the practice of fgm .
the commonest basis would be the ancient and tradition beliefs [ 20 , 21 ] .
these observations are similar to those in data from burkina faso , mali , guinea , and gambia in west africa . about the clinical findings , fgm types
i and ii accounted for 100% of cases whereas in somalia , sudan and egypt , mali , and burkina faso types iii and iv were mentioned up to 89% [ 2325 ] .
the immediate complications , such as pain , fever , and minimal or incidental bleeding as short period of bleeding ( if it is severe ) could be catastrophic , were probably underestimated .
hemorrhages , infections , and death have been reported together with the posttraumatic stress disorders and memory problems [ 26 , 27 ] . what is the future of our patients ?
most did not have major long - term complications , after the fear and the psychological trauma of fgm , finding similar to those of althaus .
these infants and young girls , once adults , could nonetheless face the late consequences of fgm .
painful intercourse , bleeding , dystocia , long labor , and episiotomy needed at the time of labor have been reported by gynecologists and many nongovernmental organizations fighting for the abandonment of fgm [ 29 , 30 ] .
although in our study no major psychological troubles were encountered , posttraumatic stress disorders have been reported in patients similar to ours [ 3135 ] .
many african countries and elsewhere in the world have laws prohibiting fgm . nongovernmental organizations ( ngos ) campaigns against fgm continue to fight for its abandonment [ 3640 ] . despite these laws and campaigns to eliminate it
, fgm continues in urban and rural areas , as our study and other recent reports have shown [ 41 , 42 ] .
women , having a past history of fgm , play the key role in the occurrence of fgm in their daughters . only types i and ii mutilations have been met .
the combination of law - enforcement together with information and education activities by ngo 's aimed at female populations has curtailed fgm in most countries .
continuing efforts are needed to eliminate fgm as a threat to the health and wellbeing of women . | the practice of female genital mutilations continues to be recurrent in african communities despite the campaigns , fights , and laws to ban it .
a survey was carried out in infants and young girls at the general hospital of abobo in cote d'ivoire .
the purpose of the study was to describe the epidemiological aspects and clinical findings related to fgm in young patients .
four hundred nine ( 409 ) females aged from 1 to 12 years and their mothers entered the study after their consent .
the results were that 60/409 patients ( 15% ) were cut .
the majority of the young females came from muslim families ( 97% ) ; the earlier age at fgm procedure in patients is less than 5 years : 87% . amongst 409 mothers , 250 women underwent fgm which had other daughters cut .
women were mainly involved in the fgm and their motivations were virginity , chastity , body cleanliness , and fear of clitoris similar to penis . only who types i and ii were met .
if there were no incidental events occurred at the time of the procedure , the obstetrical future of these young females would be compromised . with fgm being a harmful practice
, health professionals and ngos must unite their efforts in people education to abandon the procedure . |
left ventricular assist devices ( lvads ) have increasingly become part of the arsenal in the treatment of end - stage heart failure [ 13 ] . despite advances in pump technology , thromboembolic events and acute pump thrombosis remain potentially life - threatening complications [ 46 ] .
the clinical presentation varies from acute malfunction of the pump with heart failure , arrhythmias and/or to systemic thromboembolic events .
rates of thromboembolic events , including ischaemic stroke and acute pump thrombosis , vary between 1 and 14 % among different studies of continuous flow ( cf ) lvads with either axial or centrifugal flow [ 6 , 815 ] . recently , starling et al . reported an increasing rate of thoratec heartmate ii pump thrombosis , which was preceded by increasing lactate dehydrogenase ( ldh ) , and was associated with substantial morbidity and mortality .
other reports showed a strong association of haemolysis with increased pump power and with partial or complete lvad thrombosis [ 17 , 18 ] .
the current guidelines of the international society for heart and lung transplantation ( ishlt ) advise to follow up haemolysis as a sign of thrombosis .
haemolysis in the presence of altered pump function should prompt admission for optimisation of anticoagulation and antiplatelet management and possible pump exchange . however , there is no advice in the guidelines about detection of thrombosis . clinically obvious
haemolysis could be seen as dark urine , anaemia , jaundice , and/or as elevated ldh .
early detection of a thromboembolic event / pump thrombosis could help in the proper management of these lvad patients .
as thromboembolic events and pump thrombosis are part of the same disease spectrum , we sought to analyse the determinants of thromboembolic events and/or pump thrombosis in the cohort of patients with a cf - lvad implanted at our institution .
forty consecutive patients implanted with axial type continuous - flow heartmate ii lvads ( thoratec corporation , pleasanton , california ) in our institution , a tertiary referral centre for end - stage heart failure and heart transplantation , between december 2006 and july 2013 , were included in this study .
all data from lvad recipients were stored electronically in the hospital electronic patient records . according to dutch law ,
all data were readily available in the medical records of the patients and were obtained during routine treatment .
clinical events such as signs of haemolysis , heart failure or infections were examined and confirmed independently by two cardiologists ( sa , kc ) .
clinical data , ecg , laboratory and echocardiography were collected every 23 months or more frequently according to the clinical need .
likewise , lvad interrogation was performed regularly at every outpatient clinic visit by an lvad technician .
the last 12-lead ecgs before lvad implantation and at follow - up / events were analysed including rhythm , qrs width and qtc interval .
blood samples were collected serially to assess parameters of haemolysis , kidney and liver function as well as inflammation ( table 1 ) .
the treating cardiologist made the choice of and changes in medications including heart failure and antiarrhythmic drugs .
twenty - five of 40 patients ( 63 % ) already had an implantable cardioverter defibrillator according to the current guidelines .
table 1baseline characteristics of all patients with or without thromboembolic eventstotal population ( n = 40)thromboembolic event or pump thrombosis ( n = 8)no thromboembolic event or pump thrombosis ( n = 32 )
p - value
demographics
age at implantation , years46 [ 4157]56 [ 4858]45 [ 3955]0.12male gender26 ( 65)6 ( 75)20 ( 63)0.69weight , kg71 1375 1270 130.41bsa , m
1.87 0.221.92
0.191.86 0.220.44bmi , kg / m
22.5 3.023.3 2.922.3 3.00.44
aetiology
non - ischaemic cardiomyopathy23 ( 57)4 ( 50)19 ( 59)0.70 ischaemic cardiomyopathy17 ( 43)4 ( 50)13 ( 41)0.70
comorbidity
diabetes mellitus3 ( 8)03 ( 9)1.0 hypertension4 ( 10)1 ( 13)3 ( 9)1.0 previous cardiac surgery3 ( 8)2 ( 25)1 ( 3)0.10 previous pci15 ( 38)2 ( 25)13 ( 41)0.69 previous tia / cva2 ( 5)02 ( 6)1.0
intermacs class
2.4 1.02.8 1.32.3 0.90.28 i10 ( 25)2 ( 25)8 ( 25)1.0 ii9 ( 23)1 ( 12.5)8 ( 25)0.66 iii16 ( 40)2 ( 25)14 ( 44)0.44 iv5 ( 13)3 ( 38)2 ( 6)0.05inotropic support35 ( 87.5)5 ( 63)30 ( 94)0.05extra - corporal circulatory support9 ( 23)2 ( 25)7 ( 22)1.0intra - aortic balloon pump13 ( 33)013 ( 41)0.04
lvad parameters at discharge
pump speed , rpm9325 5169375 2259313 5680.76 pump flow , l / m4.9 1.24.8 1.05.0 1.20.67 pulse index4.8 0.94.9 0.74.8 0.90.92 pump power , watts6.0 1.36.0 1.06.0 1.30.94
electrocardiography
atrial fibrillation3 ( 8)03 ( 9)1.0 qrs duration , ms146 71160 54143 750.56 qtc , ms462 49506 35451 460.003
echocardiography
left atrial dimensions , mm48 1251 947 120.51 lv end - diastolic dimension , mm67 1663 1466 170.46 lv end - systolic dimension , mm61 1663 1461 170.76
baseline laboratory values
lactate dehydrogenase , u / l407 [ 321849]361 [ 277455]433 [ 3331101]0.21 nt - probnp , pmol / l1136 1112800 4711222 12160.35 total bilirubin , umol / l24 2120 1425 230.52 bun , mmol / l16 1023 1714 70.03 creatinine , umol / l147 89143 65148 950.89 crp mg / l56
7144 5459 750.56 alat , u / l314 684126 227361 7520.39 asat , u / l297 636149 268334 6960.47 albumin , g / l30 629 430 70.66 haemoglobin , mmol / l7.1 1.27.5 1.47.0 1.10.26 haematocrit l / l0.35 0.060.38 0.080.34 0.060.19 wbc count , 1000/mm
10.1 5.49.3 2.710.3 5.90.63 platelet count , 1000/mm
207 89250 97196 850.13categorical variables are presented as frequencies and percentages .
continuous variables are presented as mean standard deviation or median [ iqr ( interquartile range 25th , 75th percentile ) ] .
iqr interquartile range , bsa body surface area , bmi body mass index , pci percutaneous coronary intervention , tia transient ischaemic attack , cva ischaemic cerebrovascular accident , interagency registry for mechanically assisted circulatory support ( intermacs ) , lvad left ventricular assist device , rpm revolutions per minute , nt - pro - bnp n - terminal of the prohormone brain natriuretic peptide , bun blood urea nitrogen , crp c - reactive protein , alt alanine aminotransferase , ast aspartate aminotransferase , wbc white blood cell .
baseline characteristics of all patients with or without thromboembolic events categorical variables are presented as frequencies and percentages .
continuous variables are presented as mean standard deviation or median [ iqr ( interquartile range 25th , 75th percentile ) ] .
iqr interquartile range , bsa body surface area , bmi body mass index , pci percutaneous coronary intervention , tia transient ischaemic attack , cva ischaemic cerebrovascular accident , interagency registry for mechanically assisted circulatory support ( intermacs ) , lvad left ventricular assist device , rpm revolutions per minute , nt - pro - bnp n - terminal of the prohormone brain natriuretic peptide , bun blood urea nitrogen , crp c - reactive protein , alt alanine aminotransferase , ast aspartate aminotransferase , wbc white blood cell . according to the ishlt guidelines
on postoperative day 12 , intravenous heparin was started if there was no evidence of bleeding . on day 25 , after removal of the chest tubes , aspirin 80 mg daily and vitamin k antagonists were started with a target international normalised ratio ( inr ) of 2.02.5 . in case of a suspected thromboembolic event / pump thrombosis , intravenous heparin was started along with clopidogrel 75 mg / day .
the target inr was increased to 2.53.5 or 3.04.0 in case of asymptomatic ( laboratory only ) signs of haemolysis versus thromboembolic event / pump thrombosis , respectively . in case of acute pump thrombosis , thrombolytic therapy ( alteplase : bolus 15 mg in 12
50 mg ) continuous infusion in 90 min , and 0.5 mg / kg ( max .
35 mg ) continuous infusion in the second 90 min ) was given .
pump thrombosis was defined as signs and symptoms of otherwise unexplained heart failure with signs of lvad dysfunction and haemolysis , thromboembolic events as cerebrovascular accident ( cva ) or transient ischaemic attack ( tia ) , as confirmed by a neurologist .
haemolysis was diagnosed according to the ishlt guidelines and the interagency registry of mechanically assisted circulatory support ( intermacs ) on analysis of pump thrombosis [ 7 , 20 ] .
laboratory and clinical diagnosis of lvad haemolysis and thrombosis were considered according to these ishlt guidelines and the intermacs registry . in the ishlt guidelines , screening for haemolysis is indicated in the setting of an unexpected drop in the haemoglobin or haematocrit level along with other clinical signs of haemolysis , such as haematuria .
screening for haemolysis with serum ldh , plasma free haemoglobin in addition to the haemoglobin or haematocrit level is recommended .
the intermacs recently specified a new definition , accepted by the us food and drug administration and industry , indicating a lower threshold of biochemical markers of haemolysis .
they used a cut - off value of serum free haemoglobin > 40 mg / dl in association with clinical signs of haemolysis beyond 72 h post - implantation to define haemolysis .
continuous variables are presented as mean standard deviation or median ( interquartile range 25th , 75th percentile ) .
continuous variables were compared using paired or independent t - test , mann whitney u - test or wilcoxon s test when appropriate . when comparing frequencies , the chi - square or fisher s exact test was used , where applicable .
all tests were two - tailed and p - values less than 0.05 were considered statistically significant .
all p - values between 0.05 and 0.10 were considered to be a statistical trend .
multivariate analysis was not done due to the too low number of events and small population .
all data from lvad recipients were stored electronically in the hospital electronic patient records . according to dutch law ,
all data were readily available in the medical records of the patients and were obtained during routine treatment .
clinical events such as signs of haemolysis , heart failure or infections were examined and confirmed independently by two cardiologists ( sa , kc ) .
clinical data , ecg , laboratory and echocardiography were collected every 23 months or more frequently according to the clinical need .
likewise , lvad interrogation was performed regularly at every outpatient clinic visit by an lvad technician .
the last 12-lead ecgs before lvad implantation and at follow - up / events were analysed including rhythm , qrs width and qtc interval .
blood samples were collected serially to assess parameters of haemolysis , kidney and liver function as well as inflammation ( table 1 ) .
the treating cardiologist made the choice of and changes in medications including heart failure and antiarrhythmic drugs .
twenty - five of 40 patients ( 63 % ) already had an implantable cardioverter defibrillator according to the current guidelines .
table 1baseline characteristics of all patients with or without thromboembolic eventstotal population ( n = 40)thromboembolic event or pump thrombosis ( n = 8)no thromboembolic event or pump thrombosis ( n = 32 )
p - value
demographics
age at implantation , years46 [ 4157]56 [ 4858]45 [ 3955]0.12male gender26 ( 65)6 ( 75)20 ( 63)0.69weight , kg71 1375 1270 130.41bsa , m
1.87 0.221.92
0.191.86 0.220.44bmi , kg / m
22.5 3.023.3 2.922.3 3.00.44
aetiology
non - ischaemic cardiomyopathy23 ( 57)4 ( 50)19 ( 59)0.70 ischaemic cardiomyopathy17 ( 43)4 ( 50)13 ( 41)0.70
comorbidity
diabetes mellitus3 ( 8)03 ( 9)1.0 hypertension4 ( 10)1 ( 13)3 ( 9)1.0 previous cardiac surgery3 ( 8)2 ( 25)1 ( 3)0.10 previous pci15 ( 38)2 ( 25)13 ( 41)0.69 previous tia / cva2 ( 5)02 ( 6)1.0
intermacs class
2.4 1.02.8 1.32.3 0.90.28 i10 ( 25)2 ( 25)8 ( 25)1.0 ii9 ( 23)1 ( 12.5)8 ( 25)0.66 iii16 ( 40)2 ( 25)14 ( 44)0.44 iv5 ( 13)3 ( 38)2 ( 6)0.05inotropic support35 ( 87.5)5 ( 63)30 ( 94)0.05extra - corporal circulatory support9 ( 23)2 ( 25)7 ( 22)1.0intra - aortic balloon pump13 ( 33)013 ( 41)0.04
lvad parameters at discharge
pump speed , rpm9325 5169375 2259313 5680.76 pump flow , l / m4.9 1.24.8 1.05.0 1.20.67 pulse index4.8 0.94.9 0.74.8 0.90.92 pump power , watts6.0 1.36.0 1.06.0 1.30.94
electrocardiography
atrial fibrillation3 ( 8)03 ( 9)1.0 qrs duration , ms146 71160 54143 750.56 qtc , ms462 49506 35451 460.003
echocardiography
left atrial dimensions , mm48 1251 947 120.51 lv end - diastolic dimension , mm67 1663 1466 170.46 lv end - systolic dimension , mm61 1663 1461 170.76
baseline laboratory values
lactate dehydrogenase , u / l407 [ 321849]361 [ 277455]433 [ 3331101]0.21 nt - probnp , pmol / l1136 1112800 4711222 12160.35 total bilirubin , umol / l24 2120 1425 230.52 bun , mmol / l16 1023 1714 70.03 creatinine , umol / l147 89143 65148 950.89 crp mg / l56
7144 5459 750.56 alat , u / l314 684126 227361 7520.39 asat , u / l297 636149 268334 6960.47 albumin , g / l30 629 430 70.66 haemoglobin , mmol / l7.1 1.27.5 1.47.0 1.10.26 haematocrit l / l0.35 0.060.38 0.080.34 0.060.19 wbc count , 1000/mm
10.1 5.49.3 2.710.3 5.90.63 platelet count , 1000/mm
207 89250 97196 850.13categorical variables are presented as frequencies and percentages .
continuous variables are presented as mean standard deviation or median [ iqr ( interquartile range 25th , 75th percentile ) ] .
iqr interquartile range , bsa body surface area , bmi body mass index , pci percutaneous coronary intervention , tia transient ischaemic attack , cva ischaemic cerebrovascular accident , interagency registry for mechanically assisted circulatory support ( intermacs ) , lvad left ventricular assist device , rpm revolutions per minute , nt - pro - bnp n - terminal of the prohormone brain natriuretic peptide , bun blood urea nitrogen , crp c - reactive protein , alt alanine aminotransferase , ast aspartate aminotransferase , wbc white blood cell .
baseline characteristics of all patients with or without thromboembolic events categorical variables are presented as frequencies and percentages .
continuous variables are presented as mean standard deviation or median [ iqr ( interquartile range 25th , 75th percentile ) ] .
iqr interquartile range , bsa body surface area , bmi body mass index , pci percutaneous coronary intervention , tia transient ischaemic attack , cva ischaemic cerebrovascular accident , interagency registry for mechanically assisted circulatory support ( intermacs ) , lvad left ventricular assist device , rpm revolutions per minute , nt - pro - bnp n - terminal of the prohormone brain natriuretic peptide , bun blood urea nitrogen , crp c - reactive protein , alt alanine aminotransferase , ast aspartate aminotransferase , wbc white blood cell .
according to the ishlt guidelines , postoperative anticoagulation started after lvad implantation and completed postoperative haemostasis .
on postoperative day 12 , intravenous heparin was started if there was no evidence of bleeding . on day 25 , after removal of the chest tubes , aspirin 80 mg daily and vitamin k antagonists were started with a target international normalised ratio ( inr ) of 2.02.5 . in case of a suspected thromboembolic event / pump thrombosis , intravenous heparin was started along with clopidogrel 75 mg / day .
the target inr was increased to 2.53.5 or 3.04.0 in case of asymptomatic ( laboratory only ) signs of haemolysis versus thromboembolic event / pump thrombosis , respectively . in case of acute pump thrombosis , thrombolytic therapy ( alteplase : bolus 15 mg in 12
min , followed by 0.75 mg / kg ( max . 50 mg ) continuous infusion in 90 min , and 0.5 mg / kg ( max .
35 mg ) continuous infusion in the second 90 min ) was given .
pump thrombosis was defined as signs and symptoms of otherwise unexplained heart failure with signs of lvad dysfunction and haemolysis , thromboembolic events as cerebrovascular accident ( cva ) or transient ischaemic attack ( tia ) , as confirmed by a neurologist .
haemolysis was diagnosed according to the ishlt guidelines and the interagency registry of mechanically assisted circulatory support ( intermacs ) on analysis of pump thrombosis [ 7 , 20 ] .
laboratory and clinical diagnosis of lvad haemolysis and thrombosis were considered according to these ishlt guidelines and the intermacs registry . in the ishlt guidelines , screening for haemolysis is indicated in the setting of an unexpected drop in the haemoglobin or haematocrit level along with other clinical signs of haemolysis , such as haematuria .
screening for haemolysis with serum ldh , plasma free haemoglobin in addition to the haemoglobin or haematocrit level is recommended .
the intermacs recently specified a new definition , accepted by the us food and drug administration and industry , indicating a lower threshold of biochemical markers of haemolysis .
they used a cut - off value of serum free haemoglobin > 40 mg / dl in association with clinical signs of haemolysis beyond 72 h post - implantation to define haemolysis .
continuous variables are presented as mean standard deviation or median ( interquartile range 25th , 75th percentile ) .
continuous variables were compared using paired or independent t - test , mann whitney u - test or wilcoxon s test when appropriate .
when comparing frequencies , the chi - square or fisher s exact test was used , where applicable .
all tests were two - tailed and p - values less than 0.05 were considered statistically significant .
all p - values between 0.05 and 0.10 were considered to be a statistical trend .
multivariate analysis was not done due to the too low number of events and small population .
in our single - centre lvad cohort of bridge - to - transplant patients , we found clinical features and other factors associated with thromboembolic events and acute pump thrombosis in 8 of the 40 patients .
one out of 5 patients on lvad support with heartmate ii developed this catastrophic complication during a median follow - up of approximately 18 months .
thromboembolic events / pump thrombosis occurred at a minimum of 34 days and a maximal of 649 days after implantation . demographic , clinical , laboratory , pump and echocardiography characteristics of the thrombosis and no - thrombosis groups at baseline and at follow - up
the patients were divided into two groups with and without a thromboembolic event and/or pump thrombosis .
the baseline data are from the pre - implant period , the laboratory values from the day before the operation . in all patients ,
however , in three patients lvad support ended in destination therapy ( severe cvas in two and malignancy in one ) . in one patient
table 2comparison of outcome in patients with and without acute pump thrombosis / thromboembolic events at the time of the event or last follow - uptotal population ( n = 40)thromboembolic event or pump thrombosis ( n = 8)no thromboembolic event or pump thrombosis ( n = 32 )
p - value
follow - up , days
336 [ 182808]549 [ 269856]297 [ 152806]0.39death8 ( 20)0 ( 0)8 ( 25)0.17heart transplantation18 ( 45)4 ( 38)14 ( 44)1.0on - going support12 ( 30)3 ( 38)9 ( 28)0.68lvad explantation1 ( 3)1 ( 13)0 ( 0)0.20
lvad parameters
pump speed , rpm9245 3649200 2839256 3840.70 pump flow , l / m5.1 1.05.7 1.04.9 0.90.06 pulse index4.8 1.24.1 1.55.0 1.00.05 pump power , watts6.8 1.98.2 3.06.4 1.40.02
clinical haemolysis parameters
macroscopic hemoglobinuria7 ( 18)4 ( 50)3 ( 9)0.02ldh levels > 735 u / l20 ( 50)7 ( 88)13 ( 41)0.04free hb ( > 6 indicates haemolysis)15 3433 5810 220.31infection at the time of te / pt15 ( 38)5 ( 63)10 ( 31)0.13 viral5 ( 13)1 ( 13)4 ( 13)1.0 bacterial10 ( 25)4 ( 50)6 ( 19)0.07
readmissions
surgery for driveline fracture6 ( 15)1 ( 13)5 ( 16)1.0re - admission due to hf8 ( 20)2 ( 25)6 ( 19)0.65
medications at te / pt or latest follow - up
vitamin k antagonist33 ( 83)8 ( 100)25 ( 78)0.31 aspirin30 ( 75)7 ( 88)23 ( 72)0.65 clopidogrel4 ( 10)2 ( 25)2 ( 6)0.17
electrocardiography
atrial fibrillation6 ( 15)1 ( 13)5 ( 16)0.82 qrs duration , ms at event148 47161 45145 480.40 qtc , ms at event463 75535 79445 630.001
echocardiography
grade aortic regurgitation1.0 0.91.1 1.01.0 0,90.66 grade mitral regurgitation1.2 1.11.3 0.91.2 1.20.89 lv end - diastolic dimension , mm57 1561 1456 160.42 lv end - systolic dimension , mm50 1452 1350 150.73 left atrial dimensions , mm38 1138 938 111.0
laboratory findings
ldh , u / l382 [ 331591]1548 [ 7542379]363 [ 325443 ] < 0.0001 nt - probnp , pmol / l473 890915 1551342 5580.11 total bilirubin , umol / l38 7924 1341 880.60 bun , mmol / l10 712 910 70.56 creatinine , umol / l136 166152 148132 1720.77 crp , mg / l70 9184 9267 910.64 alat , u / l82 159162 22662 1350.11 asat ,
u / l121 203185 156106 2120.33 albumin , g / l39 940 839 100.77 inr2.3 1.12.4 0.92.3 1.10.89 haemoglobin , mmol / l6.7 1.86.0 1.76.9 1.80.19 haematocrit , l / l0.33 0.090.31 0.070.34 0.090.41 wbc count , 1000/mm
10 610 610 60.99 platelet count , 1000/mm
198 84237 71188 840.14categorical variables are presented as frequencies and percentages .
continuous variables are presented as mean standard deviation or median [ iqr ( interquartile range 25th , 75th percentile ) ] .
lvad left ventricular assist device , rpm revolutions per minute , ldh lactate dehydrogenase , free hb free haemoglobin , te thromboembolic event , pt pump thrombosis , hf heart failure , lv left ventricle , nt - pro - bnp n - terminal of the prohormone brain natriuretic peptide , bun blood urea nitrogen , crp c - reactive protein , alat alanine aminotransferase , asat aspartate aminotransferase , inr international normalised ratio , wbc white blood cell .
lvad parameters at event or latest follow - up ; values presented as mean ( sd ) , median ( interquartile range ) , or n ( % ) .
comparison of outcome in patients with and without acute pump thrombosis / thromboembolic events at the time of the event or last follow - up categorical variables are presented as frequencies and percentages .
continuous variables are presented as mean standard deviation or median [ iqr ( interquartile range 25th , 75th percentile ) ] .
lvad left ventricular assist device , rpm revolutions per minute , ldh lactate dehydrogenase , free hb free haemoglobin , te thromboembolic event , pt pump thrombosis , hf heart failure , lv left ventricle , nt - pro - bnp n - terminal of the prohormone brain natriuretic peptide , bun blood urea nitrogen , crp c - reactive protein , alat alanine aminotransferase , asat aspartate aminotransferase , inr international normalised ratio , wbc white blood cell .
lvad parameters at event or latest follow - up ; values presented as mean ( sd ) , median ( interquartile range ) , or n ( % ) . at baseline ,
an intra - aortic balloon pump was used significantly more often in the thrombosis group , and this group had a higher blood urea nitrogen .
furthermore , the thrombosis group showed a trend towards intermacs class iv ( p = 0.05 ) and less inotropic use ( p = 0.05 ) . at the last follow - up ( july 2013 ) , eight ( 20 % ) patients had one or more thromboembolic events or pump thrombosis at median follow - up of 549 [ 269856 ] days : tia in 4 patients , ischaemic cva in 3 patients and acute pump thrombosis in 2 patients .
there was no difference in survival between the groups censored for heart transplantation or lvad explantation ( p = 0.13 , fig . 1 ) .
one patient with pump thrombosis was treated with acute pump replacement ( table 3 and fig .
four patients in the no - thrombosis group had a non - ischaemic cva , one due to an air embolism and three due to intracerebral bleeding .
in these patients , a neurologist ruled out an ischaemic stroke by ct scan . at the time of the thromboembolic event / pump thrombosis , higher pump power was seen in thrombosis group compared with the no - thrombosis group ( 8.2 3.0 vs. 6.4 1.4 w , p = 0.02 ) , as well as a trend towards a lower pulse index ( 4.1 1.5 vs. 5.0 1.0 p = 0.05 ) and a trend towards higher pump flow ( 5.7 1.0 vs. 4.9
1.9 l m p = 0.06 ) macroscopic haemoglobinuria was seen in 4 of 8 patients of the thrombosis group and 3 of 32 patients of no - thrombosis group ( 50 vs. 9 % , p = 0.02 ) .
the patients in the thrombosis group with macroscopic haemoglobinuria had concomitant infections ; at that time they had a therapeutic inr or activated partial thromboplastin time .
the presence of a thrombus in the pump was confirmed at explantation by the thoracic surgeon and manufacturer in 4 of the 8 patients .
1kaplan - meyer curve for survival during lvad support for the thrombosis group ( te / pt+ ) and the no - thrombosis group ( te / pt ) .
patients are censored at heart transplantation and lvad explantation kaplan - meyer curve for survival during lvad support for the thrombosis group ( te / pt+ ) and the no - thrombosis group ( te / pt ) .
patients are censored at heart transplantation and lvad explantation table 3detailed overview of the eight patients with thromboembolic events during follow - uppatient no.12345678age ( years)4649576437595754sexmmmmffmmaetiology heart failurecmpcmpihdihdcmpihdcmpihdintermacs class44231134total support time ( days)16036141057789483182298180time to event ( days)63117564934195718949eventtiatiacvatiacvatia + cvapump thrombosispump thrombosisinfection at the time of the eventviral upper airway infectionnonesepsis e.c.inonenoneurinary tract infectionbacterial prostatitisurinary tract infectionculturenonenone
staphyl .
species ( cns )
nonenone
enterococcus faecalis
citrobacter freundi
morganella morganii
treatment at the time of eventasa / oacasa / oacasa / oacoac
asa / oacasa / oacasa / oacasa / oacnt - probnp ( pmol / l)85483393986523345391611inr2.22.82.41.42.24.02.12.5macroscopic haematurianonononoyesyesyesyesfree - hb198350121172peak ldh ( u / l)4227452286757213196526583532target inr342.53.52.53.52.53.5clopidogrel + 2.53.5clopidogrel + 2.53.5clopidogrel + 2.53.5 + alteplaseurgent pump exchangesuccess of treatmentyesyesyesyesyesyespartlynooutcomehtxhtxlvad dthtxsuccessfully explantedhtx : deathsemi urgent htxurgent htx
cmp cardiomyopathy , ihd ischaemic heart disease , tia transient ischaemic attack , cva ischaemic cerebrovascular attack ; e.c.i ( e causa ignota ) , cns coagulase - negative staphylococci , asa aspirin , oac oral anticoagulation , inr international normalised ratio , hb haemoglobin , ldh lactate dehydrogenase , htx heart transplantation , lvad left ventricular assist device , dt destination therapy .
no aspirin due to active duodenal ulcer .
aspirin started late ( 5 months post lvad ) due the peri - hepatic haematoma .
detailed overview of the eight patients with thromboembolic events during follow - up
cmp cardiomyopathy , ihd ischaemic heart disease , tia transient ischaemic attack , cva ischaemic cerebrovascular attack ; e.c.i ( e causa ignota ) , cns coagulase - negative staphylococci , asa aspirin , oac oral anticoagulation , inr international normalised ratio , hb haemoglobin , ldh lactate dehydrogenase , htx heart transplantation , lvad left ventricular assist device , dt destination therapy .
no aspirin due to active duodenal ulcer .
aspirin started late ( 5 months post lvad ) due the peri - hepatic haematoma .
fig . 2
a explanted pump inlet rotor in a 54-year - old male ( patient no . 9 in table 3 ) with acute pump thrombosis . due to acute pump thrombosis
, the patient had acute left- and right - sided heart failure with signs of severe haemolysis and acute renal failure .
macroscopic fresh white and red old thrombus is shown on the rotor , as confirmed by the manufacturer .
b ldh course of the 57-year - old male ( patient no . 7 in table 3 ) presenting with acute pump thrombosis successfully treated with recombinant tissue - plasminogen activator ( rt - pa ) .
this patient had several episodes of an abrupt peak of ldh during therapeutic inrs associated with relapsing urinary tract infections ( citrobacter freundii ) .
at the highest ldh peak he developed acute pump thrombosis , which was treated with thrombolytic therapy ( alteplase ) . dashed line = upper limit of normal value ldh .
c time course of serum ldh ( u / l ) in a 37-year - old woman ( patient no . 5 in table 3 ) , 6 months on lvad support , admitted with a ischaemic cerebrovascular event and response to various therapeutic interventions .
cva cerebrovascular accident , inr international normalised ratio , asa acetylsalicylic acid , iv intravenous . dashed line = upper limit of normal value ldh
a explanted pump inlet rotor in a 54-year - old male ( patient no . 9 in table
, the patient had acute left- and right - sided heart failure with signs of severe haemolysis and acute renal failure .
macroscopic fresh white and red old thrombus is shown on the rotor , as confirmed by the manufacturer .
b ldh course of the 57-year - old male ( patient no . 7 in table 3 ) presenting with acute pump thrombosis successfully treated with recombinant tissue - plasminogen activator ( rt - pa ) .
this patient had several episodes of an abrupt peak of ldh during therapeutic inrs associated with relapsing urinary tract infections ( citrobacter freundii ) . at the highest ldh peak he developed acute pump thrombosis , which was treated with thrombolytic therapy ( alteplase ) .
c time course of serum ldh ( u / l ) in a 37-year - old woman ( patient no . 5 in table 3 ) , 6 months on lvad support , admitted with a ischaemic cerebrovascular event and response to various therapeutic interventions .
cva cerebrovascular accident , inr international normalised ratio , asa acetylsalicylic acid , iv intravenous . dashed line = upper limit of normal value ldh furthermore , the thrombosis group had more than fourfold higher lactate dehydrogenase levels ( median ldh : 1548 [ iqr : 7542379 ] vs. 363 [ iqr : 325443 ] u / l , p < 0.0001 ) . in the thrombosis group ,
infection was associated with a more than threefold increase of ldh with or without clinical signs of tia / cva or pump thrombosis . in 63 % ( vs. 31 % in the no - thrombosis group , p = 0.13 ) of the patients presenting with thromboembolism / pump thrombosis , an infection ( bacterial in 4 and viral in 1 of the 8 patients ) was confirmed . at baseline and at follow - up
, there was a significantly longer corrected - qt interval in the thrombosis group . the sensitivity and specificity of ldh as a marker of haemolysis ( cut - off value three times the upper limit of normal ( ldh > 735
u / l ) were 88 and 97 % , respectively , with the positive and negative predictive value being 88 vs. 97 % , respectively .
in our single - centre lvad cohort of bridge - to - transplant patients , we studied the clinical features and associated factors in patients with thromboembolic events and acute pump thrombosis .
one out of 5 patients on lvad support with a heartmate ii developed a thromboembolic event / pump thrombosis during a median follow - up of approximately 18 months .
infection was confirmed at the time of the event in two - thirds of these patients .
elevated pump power and macroscopic haemoglobinuria predicted thromboembolic events / pump thrombosis , but ldh more than three times the upper level of normal was the best biochemical parameter in predicting and guiding the management of lvad - related thromboembolic events and/or acute pump thrombosis , with a sensitivity of 88 % and specificity of 97 % .
cf - lvads have been increasingly used in the last decade as a bridge to transplantation .
thromboembolic events / pump thrombosis remain one the most feared common complications in cf - lvad patients , although bleeding results in the most morbidity and mortality [ 2223 ] .
the anticoagulation strategy of whitson et al . has been nuanced because it requires a delicate balance of adequate anticoagulation to minimise thrombotic complications but not so excessive that it will cause bleeding complications ( e.g. , gastrointestinal or neurological bleeding ) .
what further complicates the clinical picture is the inherent haematological effects of cf - lvads [ 2425 ] . in our cohort
there were 23/40 cases of early post - implantation bleeding , 21 of which resulted in early cardiac tamponade . in their analysis in 2008 , john et al
. found a low thromboembolic risk ( 4.4 % ) in heartmate ii patients even with less stringent requirements for anticoagulation , which was confirmed by menon et al . in 2012 . from 2013
, there has been an increase in thromboembolic events / pump thrombosis in heartmate ii patients to 13.4 % , according to whitson et al . .
in our report , 5 of the 40 patients ( 12.5 % ) had a cva or pump thrombosis , if tias were excluded due their mild clinical sequelae .
acute pump thrombosis is a life - threatening condition and its optimal management requires early intervention .
detection of the earliest signs of pump thrombosis could lead to successful thrombolysis of a soft developing thrombus .
examined 177 lvad patients of whom 19 ( 11 % ) developed acute pump thrombosis , whereby all underwent pump exchange ; the recurrence rate was 1 % . in one - third of the patients , inadequate anticoagulation was found due to withholding or cessation of anticoagulation .
thrombolysis could also be used with varying effect , as in our cases in fig .
2a , b and c [ 11 , 27 ] . in our experience , the use of clopidogrel on top of aspirin and coumarins in optimising anticoagulation seems effective and safe , but the efficacy of antiplatelet therapy using clopidogrel in one study was not sufficient in more than 50 % of the patients . in a multicentre analysis
it was recently demonstrated that haemolysis causes long - term negative effects in the long - term course of lvad support . in 7 % of their 115 patients with a heartmate ii ,
hasin et al . found signs of haemolysis presenting with very high ldhs ( more than six times normal ) which after intensifying the anticoagulation therapy decreased to baseline within 2 weeks .
. a recent analysis of the intermacs registry of 4850 patients frequently found a mean time to event of 7.4 months and a cumulative incidence of 9 % at 2 years . in another cohort of 20 consecutive cases of pump thrombosis , haematological markers , including ldh , plasma free haemoglobin and creatinine , were the only reliable sign of lvad thrombosis , as opposed to echocardiographic or pump parameters .
along with the emerging association of ldh with thromboembolism in patients with heartmate ii , there is also a growing association with acute infections , probably due to increased hypercoagulability [ 15 , 31 ] .
we can confirm this in our cohort , where there seems to be a correlation between infection and thrombosis in lvad .
ldh seems to be a very powerful parameter in predicting serious thromboembolic adverse events in heartmate ii patients .
interestingly , the qtc was longer in the thrombosis group compared with the no - thrombosis group both at baseline and at latest follow - up .
it is known that qtc is a very crucial prognostic parameter in end - stage heart failure and we see here an association with the development of thrombosis . to our knowledge
this study has several limitations , which should be taken into account in the final interpretation of the data .
the design is a retrospective study , the number of cases is very limited , and there was no routine follow - up and analysis of eventual hypercoagulability and/or antiplatelet drugs resistance .
also , our findings were restricted to heartmate ii and may not be applicable to patients supported with other types of cf - lvads .
cf - lvads have been increasingly used in the last decade as a bridge to transplantation .
thromboembolic events / pump thrombosis remain one the most feared common complications in cf - lvad patients , although bleeding results in the most morbidity and mortality [ 2223 ] .
the anticoagulation strategy of whitson et al . has been nuanced because it requires a delicate balance of adequate anticoagulation to minimise thrombotic complications but not so excessive that it will cause bleeding complications ( e.g. , gastrointestinal or neurological bleeding ) .
what further complicates the clinical picture is the inherent haematological effects of cf - lvads [ 2425 ] . in our cohort
there were 23/40 cases of early post - implantation bleeding , 21 of which resulted in early cardiac tamponade . in their analysis in 2008 , john et al
. found a low thromboembolic risk ( 4.4 % ) in heartmate ii patients even with less stringent requirements for anticoagulation , which was confirmed by menon et al . in 2012 . from 2013
, there has been an increase in thromboembolic events / pump thrombosis in heartmate ii patients to 13.4 % , according to whitson et al . .
in our report , 5 of the 40 patients ( 12.5 % ) had a cva or pump thrombosis , if tias were excluded due their mild clinical sequelae .
acute pump thrombosis is a life - threatening condition and its optimal management requires early intervention .
detection of the earliest signs of pump thrombosis could lead to successful thrombolysis of a soft developing thrombus .
examined 177 lvad patients of whom 19 ( 11 % ) developed acute pump thrombosis , whereby all underwent pump exchange ; the recurrence rate was 1 % . in one - third of the patients , inadequate anticoagulation was found due to withholding or cessation of anticoagulation .
thrombolysis could also be used with varying effect , as in our cases in fig .
2a , b and c [ 11 , 27 ] . in our experience , the use of clopidogrel on top of aspirin and coumarins in optimising anticoagulation seems effective and safe , but the efficacy of antiplatelet therapy using clopidogrel in one study was not sufficient in more than 50 % of the patients .
in a multicentre analysis it was recently demonstrated that haemolysis causes long - term negative effects in the long - term course of lvad support . in 7 % of their 115 patients with a heartmate ii ,
hasin et al . found signs of haemolysis presenting with very high ldhs ( more than six times normal ) which after intensifying the anticoagulation therapy decreased to baseline within 2 weeks .
. a recent analysis of the intermacs registry of 4850 patients frequently found a mean time to event of 7.4 months and a cumulative incidence of 9 % at 2 years . in another cohort of 20 consecutive cases of pump thrombosis , haematological markers , including ldh , plasma free haemoglobin and creatinine , were the only reliable sign of lvad thrombosis , as opposed to echocardiographic or pump parameters .
along with the emerging association of ldh with thromboembolism in patients with heartmate ii , there is also a growing association with acute infections , probably due to increased hypercoagulability [ 15 , 31 ] .
we can confirm this in our cohort , where there seems to be a correlation between infection and thrombosis in lvad .
ldh seems to be a very powerful parameter in predicting serious thromboembolic adverse events in heartmate ii patients .
interestingly , the qtc was longer in the thrombosis group compared with the no - thrombosis group both at baseline and at latest follow - up .
it is known that qtc is a very crucial prognostic parameter in end - stage heart failure and we see here an association with the development of thrombosis . to our knowledge
this study has several limitations , which should be taken into account in the final interpretation of the data .
the design is a retrospective study , the number of cases is very limited , and there was no routine follow - up and analysis of eventual hypercoagulability and/or antiplatelet drugs resistance .
also , our findings were restricted to heartmate ii and may not be applicable to patients supported with other types of cf - lvads .
in patients with cf - lvad ( heartmate ii ) , thromboembolic events and/or pump thrombosis are associated with symptoms and signs of acute haemolysis as manifested by high ldh , elevated pump power and decreased pulse index , especially in the context of an infection .
these symptoms and signs could help in the early diagnosis and timely intensification of antithrombotic and/or antiplatelet therapy to prevent acute pump thrombosis and thromboembolic events or the need for pump replacement .
| backgrounddespite advances in pump technology , thromboembolic events / acute pump thrombosis remain potentially life - threatening complications in patients with continuous - flow left ventricular assist devices ( cf - lvad ) .
we sought to determine early signs of thromboembolic event / pump thrombosis in patients with cf - lvad , which could lead to earlier intervention.methodswe analysed all heartmate ii recipients ( n = 40 ) in our centre between december 2006 and july 2013 .
thromboembolic event / pump thrombosis was defined as a transient ischaemic attack ( tia ) , ischaemic cerebrovascular accident ( cva ) , or pump thrombosis.resultsduring median lvad support of 336 days [ iqr : 182808 ] , 8 ( 20 % ) patients developed a thromboembolic event / pump thrombosis ( six tia / cva , two pump thromboses ) . at the time of the thromboembolic event / pump thrombosis , significantly higher pump power was seen compared with the no - thrombosis group ( 8.2 3.0 vs. 6.4 1.4 w , p = 0.02 ) , as well as a trend towards a lower pulse index ( 4.1 1.5 vs. 5.0 1.0 , p = 0.05 ) and a trend towards higher pump flow ( 5.7 1.0 vs. 4.9 1.9 l m , p = 0.06).the thrombosis group had a more than fourfold higher lactate dehydrogenase ( ldh ) median 1548 [ iqr : 7542379 ] vs. 363 [ iqr : 325443 ] u / l , p = 0.0001 ) .
bacterial ( n = 4 ) or viral ( n = 1 ) infection was present in 5 out of 8 patients .
ldh > 735 u / l predicted thromboembolic events / pump thrombosis with a positive predictive value of 88 % .conclusionsin patients with a cf - lvad ( heartmate ii ) , thromboembolic events and/or pump thrombosis are associated with symptoms and signs of acute haemolysis as manifested by a high ldh , elevated pump power and decreased pulse index , especially in the context of an infection . |
it can negatively influence the patient 's quality of life and may cause important complications such as malnutrition , weight loss , and aspiration.1 benign esophageal strictures are caused by various disorders and procedures such as gastroesophageal reflux disease , surgery ( anastomotic stricture ) , radiotherapy , ablative therapy , caustic ingestion , and pill - induced injury.2,3 esophageal strictures can be divided into two types : simple and complex .
simple strictures are defined as short , focal , straight , and easy to pass through with a standard diagnostic endoscope , and are mostly caused by schatzki rings , esophageal webs , or peptic injury .
complex strictures are longer ( > 2 cm ) , tortuous , angulated , or have a severely narrowed diameter ( < 12 mm ) and often do not allow passage of an endoscope .
complex strictures can be mainly caused by surgery , radiotherapy , or corrosive injury.1,2 of the two types of strictures , the complex type is difficult to treat and associated with a higher recurrence rate than the simple type .
refractory strictures have the following characteristics : an anatomic fibrotic esophageal restriction , absence of inflammation or motility disorder , and inability to achieve a diameter of 14 mm in five sessions of dilatations at 2-week intervals or inability to maintain a diameter of 14 mm for 4 weeks once this diameter has been achieved.4 until now , serial endoscopic dilatation with bougies or balloons has been the standard treatment for esophageal strictures .
although the immediate success rate for relieving dysphagia is 80% to 90% , the recurrence rate reaches 30% to 40% during long - term follow - up , especially in complex strictures , requiring repeated dilatations or even surgery.5,6 what is worse , up to 10% of patients will not experience any meaningful improvement and be unresponsive to repeated endoscopic dilatations . in these cases ,
repeated sessions of dilatation without long - term clinical success are closely linked to increased physical and emotional burden to patients ; thus , an alternative treatment strategy should be introduced.7,8,9 as an alternative , surgical solutions such as gastric pull - up and enteral replacement are most effective ; however , many patients are not good surgical candidates because of their comorbid conditions or unwillingness to undergo surgery because of a substantial risk of morbidity and mortality.7,10,11 endoscopic temporary placement of a self - expanding esophageal stent has been proposed as a well - accepted and effective treatment for refractory esophageal strictures owing to its longer - lasting dilatation effects , ability to maintain luminal patency , and simultaneous stretching of the strictures in comparison with dilatation procedures ; moreover , its favorable outcomes , as well as lower morbidity and mortality than surgery have been reported.12,13,14 currently , three types of esophageal stents are available : self - expanding metal stent ( sems ) , self - expanding plastic stent ( seps ) , and biodegradable ( bd ) stent ( fig .
1).2,15 this review focuses on the current experience with esophageal stents , mainly seps and bd stents , for complex and refractory benign esophageal strictures .
seps can be easily removed and seems not to be inferior to metallic stent.16 initially , seps was found to provide significant improvement of dysphagia and quality of life , and to decrease the number of dilatation sessions in patients with benign strictures.2,17 on the basis of these results , the use of seps in patients with benign esophageal strictures has been proposed as an alternative to sems.18 the currently available seps is the polyflex stent ( boston scientific , natick , ma , usa ) , which is composed of a plastic wire and has a fully covered design with silicone and a proximal flare . this stent material has been suggested to reduce reactive tissue hyperplasia . as a result ,
the stent is easily removable and approved by food and drug administration for the treatment of benign esophageal strictures .
the use of polyflex stents in malignant strictures has shown good relief of dysphagia , a major complication rate of 9% , and a recurrent dysphagia rate of 37% , mostly due to stent migration ( 13% to 29% ) , tissue overgrowth ( 10% to 30% ) , and food impaction ( 5%).2,16,19 for benign strictures , varying results have been reported .
the initial study series in benign esophageal strictures showed promising results , with relief of dysphagia in 80% of patients.8,20,21,22 seps seems to result in a meaningful reduction of reactive tissue hyperplasia and safe stent removal owing to the fully covered design and the silicone material .
however , results of a later series have shown a high migration rate ( up to 62% ) and low long - term relief of dysphagia ( only 17% to 30% of patients).3,4,5,10 a recent systematic review of 130 patients with a benign esophageal stricture , obtained from 10 studies , showed that after a median follow - up period of 13 months , only 52% of the patients were dysphagia - free , the early migration rate was 24% , the endoscopic reintervention rate was 21% , and major clinical complications occurred in 9% of patients.3 our systematic review of 172 patients with a benign esophageal stricture , including recent updated reports at the time of writing of this review article , shows the following results : technical success rate of 98% , clinical success rate of 45% , and early stent migration rate of 31% ( table 1).10,12,17,20,23,24,25,26,27,28,29,30
considering that recent studies on seps have shown less favorable outcomes , including a disappointing long - term efficacy , high migration rate , and frequent needs for reintervention , the bd stent has been developed as an alternative to seps .
the currently available bd stent designs are the ella - bd stent ( ella - cs , hradec kralove , czech republic ) , which is composed of polydioxanone , a surgical suture material,31 and the poly - l - lactic acid ( plla)-bd stent ( marui textile machinery , osaka , japan ) , which consists of knitted plla monofilaments ( table 2).32 these stents can be degraded by hydrolysis , which is accelerated at low ambient ph .
generally , stents begin to degrade after 4 to 5 weeks and dissolve during a period of 2 to 3 months .
the major strength of bd stent over sems or seps is that it does not require removal , even when migrated , and it can be left until it is dissolved by gastric acid , which accelerates hydrolysis , thus avoiding further procedures and potential morbidity.31 subsequent studies have shown the availability of the plla - bd stent , with a low stent - related complication rate , compared with sems and seps.32,33,34 in these studies , there were no symptoms or need for reintervention within the follow - up period in all cases ; however , most of the stents ( 10 of 13 cases [ 70% ] ) had migrated within 10 to 21 days after insertion .
because of this tendency of early stent migration found in those three studies , the natural history of degradation of the plla - bd stent within the esophagus and the tolerability of the degradation process over time were not adequately assessed . in contrast to the plla - bd stent , the ella - bd stent has shown some promising results .
an initial case series with the ella - bd stent for esophageal strictures , including caustic , peptic , malignant , anastomotic , radiation - induced strictures as well as achalasia , showed a low migration rate ( 0% to 22% ) and an acceptable clinical success rate ( 33% to 60%).21,31,35,36,37 however , the ella - bd stent , not unlike commercially available sems and seps , can induce significant hyperplastic tissue reactions.31,35,36,37,38 the uncovered design of bd stent allows stent embedding to the esophageal wall and significantly reduces the migration rate ; however , it induces considerable reactive tissue hyperplasia .
our systematic review of 111 patients with a benign esophageal stricture obtained from nine studies showed the following results : technical success rate of 96% , clinical success rate of 47% , early stent migration rate of 21% , and tissue hyperplasia rate of 13% ( table 3).12,21,30,32,33,34,35,39,40 recently modified ella - bd stents with a non - bd covering made of polyurethane were used in five patients with esophageal leaks or perforations.41 the initial clinical success rate was 80% ; however , the stent migration rate was 60% .
therefore , the effect of the covered design on minimizing the hyperplastic tissue reaction remains an unsolved problem .
recently , drug - eluting bd stents have been introduced as a solution to reactive hyperplasia .
theoretically , localized delivery of drugs such as paclitaxel or rapamycin from drug - eluting stents is a promising treatment method for preventing restenosis or inflammatory cell proliferation . until now ,
many experimental studies have been done with animal models.42,43 more studies on the clinical application of drug - eluting bd stents for human patients are needed .
the main limitations of stent placement in benign strictures are hyperplastic tissue reaction , embedding into the esophageal wall , and stent migration . to avoid these limitations ,
an optimal stent must have a flexible , nontraumatic design and a diameter large enough for normal food passage .
in addition , the procedure for insertion , repositioning , and removal should be simple and minimize stent migration and tissue hyperplasia.2 however , for the treatment of complex and refractory benign esophageal strictures , no ideal stent is yet available .
each type of stent has its own advantages and disadvantages according to the stent characteristics ( table 4 ) .
to date , only uncontrolled studies have evaluated the safety and efficacy of various stent designs in patients with benign esophageal strictures . on the basis of the studies published thus far ,
the long - term outcome according to the stent type is not different and is unsatisfactory ( approximately 45% ) ( table 5 ) .
as previously mentioned , although a partially covered sems has a high anchoring capacity owing to the uncovered design at both ends , it shows a high rate of hyperplastic ingrowth or overgrowth through the uncovered mesh , resulting in severe stent embedding and difficulty of safe removal endoscopically . on the other hand ,
the migration rate is higher when fully covered stents , either sems or seps , are used .
meanwhile , the bd stent shows reduced migration rate owing to its uncovered design ; however , hyperplastic tissue reactions have emerged as a main problem . up to now ,
the question of which type of stent should be recommended for the effective treatment of complex and refractory benign strictures has no clear answer .
therefore , the selection of stent type for endoscopic treatment should be individualized , taking into consideration the endoscopist 's experience as well as patient and stricture characteristics , especially including the location and cause of the stricture . in the case of sites with an increased risk of migration ( i.e. , distal esophagus or anastomosis site ) or restenting after stent migration , the bd stent may be a reasonable option over sems or seps . to minimize tissue hyperplasia after bd stent placement , steroid injection or a drug - eluting bd stent
larger , randomized , prospective , well - designed studies are needed to demonstrate the long - term efficacy and safety of stents at each applicable location within the gastrointestinal tract .
seps and bd stents were developed to decrease the limitations of sems for benign complex and refractory esophageal strictures .
early studies on both stents reported excellent results ; however , our systematic review does not show a superior clinical outcome of these stents compared with sems .
although the ideal stent has not yet been developed , seps and bd stents have their own special merits such as decreasing tissue hyperplasia and eliminating the need for stent removal .
therefore , the selection of stent type should be individualized , taking into consideration the endoscopist 's experience as well as patient and stricture characteristics . in the future ,
a new stent that has only the merits of sems , seps , and bd stents will need to be developed . | endoscopic stent placement is a well - accepted and effective alternative treatment modality for complex and refractory esophageal strictures . among the currently available types of stents , the partially covered self - expanding metal stent ( sems )
has a firm anchoring effect , preventing stent migration and ensuring effective covering of a narrowed segment .
however , hyperplastic tissue reaction driven by the uncovered mesh may prevent easy and safe stent removal . as an alternative ,
a fully covered sems decreases the recurrence of dysphagia caused by hyperplastic tissue ingrowth ; however , it has a high migration rate .
likewise , although a self - expanding plastic stent ( seps ) reduces reactive hyperplasia , the long - term outcome is disappointing because of the high rate of stent migration .
a biodegradable stent has the main benefit of not requiring stent removal in comparison with sems and seps .
however , it still has a somewhat high rate of hyperplastic reaction , and the long - term outcome does not satisfy expectations . up to now , the question of which type of stent should be recommended for the effective treatment of complex and refractory benign strictures has no clear answer .
therefore , the selection of stent type for endoscopic treatment should be individualized , taking into consideration the endoscopist 's experience as well as patient and stricture characteristics . |
hypospadias is the second most prevalent congenital anomaly , affecting approximately 3.1 per 1,000 male live births ( 1 , 2 ) . the most common complications after hypospadias repair include urethrocutaneous fistula ( ucf ) , narrow neourethra , meatal stenosis , urethral strictures , and residual chordee ( 3 ) .
ucf , as the most common unfavorable complication of hypospadias surgery , has been found to be associated with the type and location of hypospadias , as well as with the type of hypospadias repair surgery ( 4 ) .
based on the severity of hypospadias , surgical technique , and experience of the operating surgeon , the incidence of ucf ranges from 5% to 10% ( 5 ) .
soft - tissue availability for multilayer repair ; the time duration after hypospadias repair ; the patient s age ; the fistula number , location , and size ; previous fistula repairs ; the type of suture material ; and the operating surgeon s skill are the key determinants of successful ucf repairs ( 6 , 7 ) .
many methods have been developed for ucf correction , and the best technique for fistula repair is determined based on the size , location , and number of fistulas , as well as the status of the surrounding skin .
smaller fistulas can be easily managed with simple closures ; however , a local skin flap is necessary if the fistula is larger and surrounded by vascularized skin ( 8) . among all of the available procedures , the most acceptable method for ucf closure is multilayered repair with a waterproofing layer , as these results in fewer recurrences and complications compared to other methods .
however , although the multilayer closure method has a low recurrence rate , it is time - consuming and difficult to learn , and the ucf may become larger during the tract excision .
thus , many attempts have been made to develop alternative methods using a simple procedure , with a lower risk of tract enlargement by tract excision and greater cosmetic acceptability .
in this study , we introduced a simple method for ucf repair , which can be described as release of the fistula from the surrounding tissues , with subsequent closure in two layers .
this clinical study was conducted during 2004 - 2014 at nemazi and shahid faghihi hospitals ( shiraz , iran ) on boys diagnosed with proximal , mid - shaft , or distal ucfs with a caliber of 4 mm , which had developed after primary tip repair for hypospadias .
our exclusion criteria were a fistula size of > 4 mm , meatal stenosis , urethral diverticulum and stricture , and multiple urethral fistulas requiring total breakdown for the repair .
the patients parents were informed about the surgical process , with clarification of the potential benefits and risks .
the parents were required to provide informed written consent prior to their children s participation in the study .
the patients were prepared and draped , then placed under general anesthesia in the supine position . for confirmation and identification of the fistula location and for the exclusion of any additional fistulas , sterile ophthalmic gentamycin ointment was injected into the urethra with simultaneous pressure on the bulbous urethra .
after insertion of a urethral catheter ( french 6 or 8) , the skin was incised around the site of the fistula .
after the fistula was released from the surrounding tissue and the dartos fascia , it was ligated with 5 - 0 pds sutures .
closure of the dartos fascia , as a second layer , was performed with 5 - 0 pds thread , using the continuous suture method . to confirm the complete closure of the fistula tract , after suturing of the dartos fascia
, a sterile saline irrigation solution was injected into the urethra with concurrent pressure on the bulbous urethra . for the last step ,
all patients underwent the same anesthesia and surgical procedures , which were performed by the same urological surgeon .
the success of the surgery was determined based on fistula relapse or the occurrence of other complications during the follow - up interval .
the mean age of the patients was 5.7 ( 3.10 ) years ( range 2 - 13 , median 5 ) .
thirty - seven patients had a single fistula , two patients had two fistulas , and one patient had three fistulas .
five patients had a history of previous fistula repair , one with a proximal fistula and the remaining four with distal fistulas .
the mean interval between primary tip urethroplasty and the present attempt at fistula repair was approximately 7.9 ( 2.99 ) months ( median 8 months ) .
the mean duration of the operation was 30.25 ( 5.18 ) minutes ( range 25 - 40 ; median 30 ) .
the average length of hospital stay was 1.28 ( 0.45 ) days ( range 1 - 2 ; median 1 ) .
after six months , relapse had occurred in only one patient , a six - year - old boy with a single 4-mm distal fistula , who had had no previous fistula repairs .
therefore , in 97.5% of the cases , relapse was non - existent . other complications , such as urethral stenosis , intraurethral obstruction , urinary retention , difficulty voiding , and epidermal inclusion cysts , were not seen in the other patients during the six - month follow - up period .
the results indicated that this simple technique has a high success rate for repairing ucfs measuring < 4 mm in any location .
attempts to achieve a higher success rate for closure of ucfs have led to the development of various surgical techniques , including simple closure , the trapdoor technique , an envelope - like closure , multilayered repair , and tip urethroplasty .
a previous study described some principles for the ideal repair technique : gentle tissue - handling , urethral mucosa inversion after excision of the epithelialized fistula tract , multilayer repair with a waterproofing layer , non - overlapping sutures , the use of absorbable and thin suture materials , a tension - free closure , the use of optical magnification , and needle - point cautery for coagulation ( 9 ) .
studied the effects of the size , location , and number of fistulas and the surrounding tissue on selecting the procedure and its outcome .
first , they excised the fistula tract , and smaller fistulas were repaired using the simple closure technique and subcutaneous tissue flaps . for larger fistulas with good surrounding skin , following simple closure of the fistula site , the skin was closed with a layered closure . larger multiple or recurrent fistulas with surrounding scar tissue were closed with an additional waterproofing flap .
a comparison of the success rate of the different fistula repair methods revealed that closure with a waterproofing layer was the most successful ( 100% ) , followed by layered ( 89% ) and simple ( 77% ) closures .
these rates decreased on the second attempts at simple closure ( 33% ) , but there was 100% success for closure with a waterproofing layer .
the significantly improved success rate with the addition of a waterproofing layer suggests that the use of this interposition layer can prevent the recurrence of ucfs ( 8) . some studies have shown that simple closure of ucfs has a high failure rate compared to other techniques . in a study by guan et al .
, modified prepuce - degloving repair for ucfs , compared to simple closure or y - v plasty , was shown to have a significantly lower long - term recurrence rate , supporting its wide clinical application ( 10 ) .
another study evaluating the success rate of the prepuce - degloving method versus simple closure or y - v plasty concluded that the former ( 96.2% success ) compared to the latter ( 78.7% success ) was a good choice for fistula repair after hypospadias surgery ( 11 ) .
some authors have reported that multilayer closure with an intermediate waterproofing layer between the neourethra and the skin plays a major role in decreasing the incidence of ucf recurrence , regardless of the surgical repair method .
various vascularized flaps , such as tunica vaginalis , dartos fascia , de - epithelialized skin , and buck s fascia , can be applied as the intermediate layer ( 12 - 14 ) . on the other hand ,
some studies have shown that simple closure of the ucf followed by a second - layer closure has a high success rate compared to other methods .
suggested the patio technique ( which preserves the tract and turns it inside - out ) for managing solitary ucfs following primary hypospadias repair .
this repairs the fistula by inversion of the fistula tract to the urethra , and this method is effective in treating solitary ucfs measuring < 4 mm ( 15 ) .
in contrast to the patio method , we did not invert the fistula tract , in order to reduce the risk of urethral stenosis and stricture . in a method used by jamal et al . for the management of primary small ucfs ( < 2 mm ) , after dissection of the tract
, it was ligated tightly with 5 - 0 vicryl and then the epithelium of the dissected tract was fulgurated by the diathermy . in order to prevent inclusion cyst formation ,
they concluded that this method was effective and successful in treating small - caliber ucfs ( 16 ) . in the present study , although we did not fulgurate the dissected tract , no inclusion cysts formed .
another difference between our method and jamal s was that we operated on fistulas measuring > 2 mm and included three boys with recurrent fistulas .
used the midline relaxing incision for large fistulas , then covered it with a vascularized dartos - based or tunica vaginalis flap .
this method demonstrated a high success rate for repairing midline and proximal urethral fistulas ( 17 ) . in the posterior urethral incision technique , after dissection of the skin edge around the urethral fistula and trimming the fistula edge , a posterior midline incision was made on the urethral wall ; the edge was then closed in two layers , the urethral edge and the dartos fascia .
( 18 ) . although we did not incise the ucfs , no recurrences occurred in our cases after six months of follow - up , the surgical sites showed no tension , and there were good cosmetic results .
the results showed that our simple method had a high success rate ( 97.5% ) for repairing small ( 2 mm ) and large ( 2 - 4 mm ) ucfs .
our method is equal to previous methods in terms of success rate , and it involves a short operating time and an easy learning curve .
we suggest conducting a large , randomized , controlled clinical trial with a longer follow - up period to confirm the results of the present study .
further studies should also be conducted on the success rate in patients with fistulas larger than 4 mm or who have undergone another type of primary hypospadias repair surgery .
the present method , which is simple , rapid , and easily learned , has a high success rate in small ( 2 mm ) and large ( 2 - 4 mm ) ucfs that develop after tip repair of hypospadias in any part of the penis .
relapses and complications were almost non - existent during six months of follow - up .
the present method , which is simple , rapid , and easily learned , has a high success rate in small ( 2 mm ) and large ( 2 - 4 mm ) ucfs that develop after tip repair of hypospadias in any part of the penis .
relapses and complications were almost non - existent during six months of follow - up . | backgroundurethrocutaneous fistula ( ucf ) is the most prevalent complication after hypospadias repair surgery .
many methods have been developed for ucf correction , and the best technique for ucf repair is determined based on the size , location , and number of fistulas , as well as the status of the surrounding skin.objectivesin this study , we introduced and evaluated a simple method for ucf correction after tubularized incised plate ( tip ) repair.methodsthis clinical study was conducted on children with ucfs 4 mm that developed after tip surgery for hypospadias repair .
the skin was incised around the fistula and the tract was released from the surrounding tissues and the dartos fascia , then ligated with 5 - 0 polydioxanone ( pds ) sutures .
the dartos fascia , as the second layer , was covered on the fistula tract with pds thread ( gauge 5 - 0 ) by the continuous suture method .
the skin was closed with 6 - 0 vicryl sutures .
after six months of follow - up , surgical outcomes were evaluated based on fistula relapse and other complications.resultsafter six months , relapse occurred in only one patient , a six - year - old boy with a single 4-mm distal opening , who had undergone no previous fistula repairs .
therefore , in 97.5% of the cases , relapse was non - existent .
other complications , such as urethral stenosis , intraurethral obstruction , and epidermal inclusion cysts , were not seen in the other patients during the six - month follow - up period.conclusionsthis repair method , which is simple , rapid , and easily learned , is highly applicable , with a high success rate for the closure of ucfs measuring up to 4 mm in any location . |
one of the main features of diabetes is the elevation of blood sugar with its deleterious consequences in a variety of tissues .
thus , control of the plasma glucose level is of utmost importance in the treatment of this disease . in recent years
, the idea that affecting glucose absorption in the intestine and/or the glucose reabsorption in the kidney might be a possible way to control the sugar level has evolved .
diabetes comprises a group of metabolic disorders characterised by chronic hyperglycaemia with disorders in the metabolism of carbohydrate , fat , and protein that result in defects in secretion and action of insulin .
dysfunction and failure of various organs , especially the eyes , kidneys , nerves , and heart , and the blood vessels are the usual complications of diabetes [ 3 , 4 ] .
diabetes is mainly divided into four main types including insulin - dependent diabetes mellitus ( type 1 ) , non - insulin - dependent diabetes mellitus ( type 2 ) , gestational diabetes , and other specific types .
diabetes mellitus type 2 ( t2 dm ) accounts for almost 90% of diabetes cases , with the property of insulin resistance and beta - cell dysfunction that induces hyperglycemia .
medical complications associated with t2 dm include cardiovascular disease , stroke , nephropathy , retinopathy , renal failure , and amputations of the extremities . in recent years
, much attention has been given to sodium - dependent glucose cotransporters ( sglts ) , mediators of reabsorption of glucose in the human body .
sodium - dependent glucose cotransporter 2 ( sglt2 ) is a high - capacity , low - affinity transporter expressed selectively in the s1 domain of the proximal tubule in the kidney and is responsible for 90% of renal glucose reuptake .
sodium dependent glucose cotransporter 1 ( sglt1 ) , on the other hand , is a low - capacity , high - affinity transporter distributed in the kidney , gut , and other tissues , responsible for the remaining 10% of glucose reuptake .
na - glucose cotransporter ( sglt ) is a membrane protein that plays an important role in the reabsorption of glucose in the kidneys .
sodium - dependent glucose cotransporters ( sglts ) , mediators of reabsorption of glucose in the kidney , have recently emerged as novel drug targets for the treatment of diabetes .
sglt is known to have three isoforms ( sglt1 , sglt2 , and sglt3 ) [ 1012 ] .
sglt1 is expressed primarily in the brush border membrane of mature enterocytes in the small intestine , where it absorbs dietary glucose and galactose from the gut lumen .
sglt2 , expressed exclusively in the kidney , is located in the s1 segment of the proximal convoluted tubule of the kidney .
it is a low - affinity , high - capacity cotransporter and is responsible for 90% of renal glucose reabsorption .
several therapeutic agents are available for monotherapy or for combination therapy with different mechanisms to treat diabetics , such as metformin , rosiglitazone , sitagliptin , acarbose , and glimepiride .
the obvious need for new approaches to treat patients with uncontrolled t2 dm has prompted continuous exploration of alternative targets involved in maintenance of glucose homeostasis .
phlorizin , 3-(benzo[b]furan-5-yl)-2,6-dihydroxy-4-methylpropiophenone-2-o--d - glucopyranoside , sergliflozin , and remogliflozin are o - glycosides and show strong inhibition of sglt2 .
they induce a glucosuric response , the result of the blockade of renal glucose reabsorption , and consequently lead to reduction of the blood glucose level and improvement of insulin sensitivity .
quantitative structure - activity relationship ( qsar ) studies can be utilized to predict eye irritation potential as an alternative in silico method , just as it has been used successfully to predict several other toxicological endpoints for some time .
hence , in continuation to our efforts [ 2267 ] in developing qsar studies for angiotensin ii at1 receptor , antitubercular agents , antimalarial activity , antimicrobial activity , antibacterial activity , cox inhibitors , and so forth . in this study
, we have taken thiophenyl derivatives for performing 2d and 3d quantitative structural - activity relationship analysis and calculations in order to understand their stereoelectronic properties .
genetic algorithm ( ga ) , simulated annealing ( sa ) , and stepwise forward - backward variable selection methods have been employed for selection of relevant descriptors .
the obtained results provide further insight into some beneficial information in structural modifications to design new potential sglt2 inhibitors .
the biological data set was chosen from a series of thirty - three thiophenyl derivatives as sglt2 inhibitors as potential antidiabetic agents reported by lee et al . .
the biological activity values [ ic50 ( nm ) ] reported in nanomolar units were converted to their molar units pic50 and subsequently used as the dependent variable for the qsar analysis . the converted to pic50 for the qsar analysis along with the structure of the compounds in the series are listed in table 1 ( marked with asterisk ) .
the test compounds were selected manually such that the structural diversity and wide range of activity in the data set were included . in this paper , a series of thiophenyl compounds with substitutions at x and r position of thiophenyl moiety are subjected to examining the relationships between structural modifications and activities against hsglt2 inhibitors with the help of qsar modeling .
all the computational studies were performed by v - life mds ( molecular design suite ) 3.5 software supplied by v - life sciences technologies pvt . ltd . ,
the sketched structures were used for the calculation of 2d molecular descriptors using qsar module of molecular design suite software .
each compound was subjected to energy minimization and batch optimization using merck molecular force field ( mmff ) , fixing root mean square gradients ( rms ) to 0.01 kcal / mol .
the sphere exclusion method was adopted total set of inhibitors was divided randomly into a training set ( 26 compounds ) for generation of qsar models and a test set ( 7 compounds ) for validation of the developed model .
this random division of data set will be done through several cycles in order to get the best qsar model .
this study will help in rational drug designing of these derivatives as sglt2 inhibitors for the eradication of t2 dm .
the maximum and minimum value in training and test set were compared in a way thatthe maximum value of pic50 of test set should be less than or equal to maximum value of pic50 of training set , the minimum value of pic50 of test set should be higher than or equal to minimum value of pic50 of training set .
the maximum value of pic50 of test set should be less than or equal to maximum value of pic50 of training set , the minimum value of pic50 of test set should be higher than or equal to minimum value of pic50 of training set . in the current approach , an attempt has been taken to understand the structural and physicochemical requirements of a set of hsglt2 inhibitors by the help of regression 2d quantitative structure - activity relationship ( 2d qsar ) .
the energy - minimized geometry was used for the calculation of the various 2d descriptors such as topological , shape and geometrical , and physicochemical parameters such as individual ( h - acceptor count , h - donor count , xlogp , retention index ( chi ) , element count , estate numbers , estate contribution , and alignment - independent descriptors were used as predictor variables ) , as they were found to be appropriate for the development of models .
a considerable number of the 265 physicochemical parameters , 300 alignment type parameters , and 99 atoms types count descriptors calculations were done using the v - life , mds 3.5 .
the preprocessing of the independent variables ( i.e. , 2d descriptors ) was done by removing the invariable ( constant column ) which resulted in a total of 216 molecular descriptors to be used for qsar analysis .
the various alignment - independent descriptors were also calculated . in this study to calculate ai descriptors ,
we have used the following attributes : 2 ( double bonded atom ) , 3 ( triple bonded atom ) , c , n , o , s , h , f , cl , br , and i , and the distance range of 07 .
the qsar models were built with the consideration of the applicability of the descriptor for the activity .
various types of physicochemical descriptors have been calculated which are shown in the data sheet ( table 3 ) .
energy minimized and geometry optimized structures of molecules were aligned by the template - based method where a template structure is defined and used as a basis for alignment of a set of molecules , and a reference molecule is chosen on which the other molecules of the data set get aligned considering the chosen template . in the present study , we aligned the database by fitting all of the compounds on most active compound 16 ( figure 1(a ) ) as an alignment template using a common substructure with the v - life mds routine database alignment .
the superimposition of all molecules based on minimizing root mean square deviation ( rmsd ) is shown in figure 1(b ) .
the steric , electrostatic , and hydrophobic fields were calculated at each lattice intersection of a regularly spaced grid of 2.0 .
methyl probe of charge + 1 with 10.0 kcal / mole electrostatic and 30.0 kcal / mole steric and hydrophobic cutoff was used for fields generation .
this resulted in calculation of 4500 field descriptors ( 1500 for each steric , electrostatic , and hydrophobic which theoretically form a continuum ) for all the compounds in separate columns ( table 3 ) .
the qsar models were assessed by the number of cross - validated r by leave - one - out method ( q ) , cross - validated standard error ( q_se ) , predicted r for external test set ( pred_r ) , and standard error for predicted r ( pred_rse ) .
the internal cross - validated predictability ( q ) was evaluated by the equation given below :
( 1)q2=1yi y^i2yiymean2 ,
where yi , yi^ are the actual and predicted activity of the ith molecule in the training set , respectively , and ymean is the average activity of all molecules in the training set .
for external validation , activity of each molecule in the test set was predicted using the model generated from the training set .
the pred_r value is calculated as follows :
( 2)pred_r2=1 yi y^i2yiymean2 ,
where yi , yi^ are the actual and predicted activity of the ith molecule in the test set , respectively , and ymean is the average activity of all molecules in the training set .
thus , the pred_r value is indicative of the predictive power of the current model based on the external test set . among several search algorithms , stepwise ( sw )
forward variable selection method , genetic algorithms ( ga ) , and simulated annealing ( sa ) based feature selection procedures are most popular for building qsar models and can explain the situation more effectively .
the models were also subjected to the test for criteria of external validation as suggested by golbraikh and tropsha . to know predictive potential of the models , squared correlation coefficient values between the observed and predicted values of the test set compounds with intercept ( r ) were calculated .
interchange of the axes gives the value of r. according to golbraikh and tropsha , models are considered acceptable , if they satisfy all of the following conditions : q >
when the observed values of the test set compounds ( y - axis ) are plotted against the predicted values of the compounds ( x - axis ) setting intercept to zero , the slope of the fitted line gives the value of k.
qsar study was performed on thiophenyl c - aryl glucoside derivatives for their sglt2 inhibitors as potential antidiabetic agents . comparison of the obtained results indicated the superiority of the genetic algorithm over the simulated annealing ( sa ) and stepwise forward - backward variable method for feature selection : pic50 = 0.6451 ( 0.1584 ) ssch3count + 0.4287 ( 0.0851 ) t_c_cl_1 0.2574 ( 0.0036 ) lumo energy + 0.2789 ( 0.0487 ) saase - index ; ntraining = 26 , ntest = 7 , degree of freedom = 21 , r = 0.8499 , q = 0.8267 , f - test = 45.8975 , r_se = 0.3098 , q_se = 0.3464 , pred_r = 0.7729 , and pred_rse = 0.6158 .
pic50 = 0.6451 ( 0.1584 ) ssch3count + 0.4287 ( 0.0851 ) t_c_cl_1 0.2574 ( 0.0036 ) lumo energy + 0.2789 ( 0.0487 ) saase - index ; ntraining = 26 , ntest = 7 , degree of freedom = 21 , r = 0.8499 , q = 0.8267 , f - test = 45.8975 , r_se = 0.3098 , q_se = 0.3464 , pred_r = 0.7729 , and pred_rse = 0.6158 . the significant model with r
= 0.8499 was considered , as model-1 showed an internal predictive power ( q = 0.8267 ) and a predictivity for the external test set ( pred_r = 0.7729 ) of about 77% . the f - test = 45.897 shows the statistical significance of 99.99% of the model which means that the probability of failure of the model is 1 in 10000 . in addition , the randomization test shows confidence of ~99.9% that the generated model is not random and hence it is chosen as the qsar model .
genetic algorithm - pls model indicates the positive contribution of ssch3count , and saase - index showed that increase in the values of these descriptors is beneficial for the sglt2 inhibitors . from the above model
, it is clear that the descriptor t_c_cl_1 contributes positively to the sglt2 inhibitors activity , which corresponds to count of number of carbon atoms separated from any chlorine atom by 1-bond distance .
thus , the presence of chloro substituents ( like in compounds 118 ) would increase the activity .
descriptor ssch3count indicates that increase in methyl group of r position may lead to an increase in the activity .
its positive value suggests that increasing the number of such carbons will lead to better sglt2 inhibitors .
the above results are in close agreement with the experimental observations , where compounds 10 , 15 , 16 , 17 , and 2833 at the r position produce sglt2 inhibitors .
molecules with negative coefficient lumo energy can accept electrons more easily than those having high lumo energy . the saase - index ( ~24% )
shows the sulphur atom connected with two aromatic bonds in the molecule and is inversely proportional to the activity .
this further suggests that the increase in electronegative atom environment adjacent to indicated sulphur atom would result in increase in the activity .
in addition , in agreement with qsar model , presence of more bulky and hydrophilic substituents like methoxy at ring r led to an increase in sglt2 potency . on the other hand ,
the residuals ( observed - predicted activity ) were found to be minimal and are presented in table 4 .
the statistical results of best model and the correlation matrix between the physicochemical parameters and the biological activity for model-1 are presented in table 5 .
also , the graph for observed versus predicted activity for the series is plotted in figure 1(d ) which shows good correlation .
molecular fields are the steric , electrostatic , and hydrophobic interaction energies which are used to develop a model for 3d qsar . in this study
, 3d qsar models were generated by knn - mfa in conjunction with genetic algorithms ( ga ) , simulated annealing ( sa ) , and stepwise ( sw ) forward - backward selection methods : pic50 = s_1044 ( 0.0317 , 0.0317 ) e_184 ( 0.2885 , 0.2885 ) s_931 ( 0.0306 , 0.0306 ) ; ntraining = 26 , ntest = 7 , q = 0.7663 , q_se = 0.3389 , f - test = 38.4683 , and pred_r = 0.7386 .
pic50 = s_1044 ( 0.0317 , 0.0317 ) e_184 ( 0.2885 , 0.2885 ) s_931 ( 0.0306 , 0.0306 ) ; ntraining = 26 , ntest = 7 , q = 0.7663 , q_se = 0.3389 , f - test = 38.4683 , and pred_r = 0.7386 .
the steric and electrostatic interaction energies are computed at the lattice points of the grid using a methyl probe of charge + 1 . the best ga - knn mfa 3d qsar model that has a q of 0.7663 and pred_r of 0.7386
the points generated in ga - knn mfa 3d qsar model are s_1044 ( 0.0317 , 0.0317 ) , e_184 ( 0.2885 , 0.2885 ) , and s_931 ( 0.0306 , 0.0306 ) .
figure 1(e ) shows the contribution plot of the three models for the electrostatic and steric fields , respectively , and indicates relative regions of the local fields around the aligned molecules leading to activity variation in the model . for electrostatic field and steric fields ,
the lattice points generated in the model are e_184 ( 0.2885 , 0.2885 ) and s_1044 ( 0.0317 , 0.0317 ) and s_931 ( 0.0306 , 0.0306 ) .
these points suggested the significance of electrostatic properties as indicated in the ranges in parentheses for maximum sglt2 inhibitory activity .
the negative value for e_184 means that electron - withdrawing substituents in this region are favorable and would increase sglt2 inhibitory activity , as shown by the presence of chlorine group in the active compounds .
therefore , less steric and more steric substituents were preferred at the position of generated data points s_1044 and s_931 , respectively , for enhancing the biological activity of thiophenyl pharmacophore .
two data points generated at the position of r around thiophenyl nucleus were steric points s 1044 and s 931 which indicates that less steric or less bulky substituents are favorable on this site .
on the other hand , less electronegative groups such as hydroxyl and nitro . the electrostatic blue ball model around r positions of the thiophenyl suggested the electron - withdrawing groups on this position benefited potency ; this may be the reason why compounds with double bonds at r positions had higher potencies than other compounds .
in addition , a red contour near the position suggested the electron - withdrawing substituent would increase the activity .
therefore , the oh at r position resulted in significant increased activity . electron - withdrawing nature of the electronegative chloro atom does contribute to the sglt2 inhibitory activity of the molecule .
the graph for observed versus predicted activity for the series is plotted in figure 1(f ) which shows good correlation .
the residuals ( observed - predicted activity ) were found to be minimal and are presented in table 4 : ntraining = 26 , ntest = 7 , q = 0.7254 , q_se = 0.3795 , f - test = 31.8965 , and pred_r = 0.6938 . ntraining = 26 , ntest = 7 , q = 0.7254 , q_se = 0.3795 , f - test = 31.8965 , and pred_r = 0.6938 .
3d data points generated , which contribute to sa knn - mfa qsar model , are shown in figure 1(g ) .
the external predictability of the above 3d qsar model using the test set was determined by pred_r , which is 0.6938 .
the points generated in sa knn - mfa 3d qsar model are e_1139 ( 0.1545 , 0.1545 ) and s_646 ( 0.3001 , 0.3001 ) , that is , electrostatic and steric interaction fields at lattice points 1139 and s_646 , respectively .
positive values for e_1139 show that electron - donating groups on the thiophenyl ring increase biological activity of compounds . on the other hand
, thiophenyl ring might be substituted with either electron - withdrawing or electron - donating groups without loss of activity .
3d qsar studies showed requirement of steric group at r position . the graph for observed versus predicted activity for the series is plotted in figure 1(h ) .
the residuals ( observed - predicted activity ) were found to be minimal and are presented in table 4 : ntraining = 26 , ntest = 7 , q = 0.7359 , q_se = 0.3562 , f - test = 30.8743 , and pred_r = 0.6743 .
ntraining = 26 , ntest = 7 , q = 0.7359 , q_se = 0.3562 , f - test = 30.8743 , and pred_r = 0.6743 .
the 3d model using the sw - knn - mfa analysis method q was found to be 0.7359 which suggests that the model could be useful for predicting sglt2 inhibitor for such thiophenyl derivatives .
these points suggested the significance and requirement of electrostatic and steric and hydrophobic properties as mentioned in the ranges for structure - activity relationship and biological activity of thiophenyl analogues .
it shows a positive contribution towards the activity , which indicates that h_1153 hydrophobic substitution at r position favors activity .
figure 1(i ) showed the hydrophobic contour the presence of a yellow contour covering the r positions of the ring indicating that hydrophobic substituents may be well tolerated in that region .
the r position of the thiophenyl was surrounded by a yellow contour which also indicated that hydrophobic groups at this position may increase activity .
less bulky substituents are tolerated at thiophenyl ring meaning that increasing size of the groups substituted in these regions reduces sglt2 inhibitory activity , since s_190 have negative values .
positive values for the hydrophobic 1153 lattice points around ring indicate that sglt2 inhibitory activity could be increased by substituting more hydrophobic groups in these regions .
similarly , the positive values of electrostatic descriptors suggested the requirement of electropositive or sterically bulky groups at the position of generated data point e_1090 around thiophenyl pharmacophore for maximum activity .
the electrostatic e_1090 data point generated which indicates sterically bulky groups such as benzene , methyl , ethyl , and isopropyl was required at r position .
the graph for observed versus predicted activity for the series is plotted in figure 1(j ) .
the residuals ( observed - predicted activity ) were found to be minimal and are presented in table 4 .
qsar study was performed on thiophenyl c - aryl glucoside derivatives for their sglt2 inhibitors as potential antidiabetic agents . genetic algorithms ( ga ) , simulated annealing ( sa ) , and stepwise ( sw ) forward - backward selection methods have been employed for selection of relevant descriptors .
comparison of the obtained results indicated the superiority of the genetic algorithm over the stepwise method for feature selection .
2d qsar further revealed that a specific group or type of descriptor is not sufficient to capture the true factors responsible for the activity in the set of inhibitor compounds .
this study also revealed that ssch3count , along with lumo energy and saase - index , forms a powerful tool to improve a qsar model .
this study used t_c_cl_1 to investigate whether a similarity based set generation method would lead to better understanding of the qsar models .
the 2d and 3d qsar suggested the presence of negative steric potential at r position in thiophenyl nucleus , that is , r position in thiophenyl nucleus should acquire less steric or less bulky substituents are favorable as well as according to models .
the constructed 3d qsar models and structure - activity relationship ( sar ) analyses of the compounds used in the study suggested that an electronegative and bulky substituent at r position and one less bulky substituent at x , r position of thiophenyl analogs are required to design novel sglt2 inhibitors .
we emphasize in this study that the e_1139 electron - donating groups on the thiophenyl ring increase biological activity of compounds .
furthermore , the knn - mfa maps offered enough information to understand the structure - activity relationship and identified structural features influencing the inhibitory activity .
the correlation of the results obtained from 3d qsar study successfully explored the primitive structure - activity relationship .
the findings can be quite useful to aid the designing of novel antidiabetic agents with high predicted potent activity . | a qsar study on thiophenyl derivatives as sglt2 inhibitors as potential antidiabetic agents was performed with thirty - three compounds .
comparison of the obtained results indicated the superiority of the genetic algorithm over the simulated annealing and stepwise forward - backward variable method for feature selection .
the best 2d qsar model showed satisfactory statistical parameters for the data set ( r2 = 0.8499 , q2 = 0.8267 , and pred_r2 = 0.7729 ) with four descriptors describing the nature of substituent groups and the environment of the substitution site .
evaluation of the model implied that electron - rich substitution position improves the inhibitory activity .
the good predictive 3d - qsar models by k - nearest neighbor ( knn ) method for molecular field analysis ( mfa ) have cross - validated coefficient q2 value of 0.7663 and predicted r2 value of 0.7386 .
the results have showed that thiophenyl groups are necessary for activity and halogen , bulky , and less bulky groups in thiophenyl nucleus enhanced the biological activity .
these studies are promising for the development of novel sglt2 inhibitor , which may have potent antidiabetic activity . |
breast cancer is on the rise , projected at a 4.5% annual increase in asr ( age standardized incident rate ) from 2006 , therefore currently approximated to be 40/100,000 from 11.7/100,000 in the 1960s .
the reasons for this steep increase are not fully established . in the recent past gene profiling in tumor tissues
suggests that breast cancers may be divided into subtypes dependent on presence or absence of oestrogen receptor , progesterone receptor , and human epidermal growth factor receptor 2 ( her 2 ) . luminal a tumors are ( er+ , pr+ ) ( er , pr+ ) , her , and luminal b tumors are ( er+ , pr+ ) , her2 + , tnbc ( er , pr ) her2 , her2+/neu ( er , pr ) her2 + .
the basal type is a subtype similar to tnbc with an overlap of 80% of identifying characteristics .
although reproductive factors have been known for decades to be associated with breast cancer risk , it is unclear to what extent these associations differ across subtypes defined by er status in sub - saharan africa populations .
literature suggests that the molecular profiles in breast tumors are generally fixed at inception ; exposures that influence the risk of developing breast cancer might be related to the tumor molecular profiles that later affect the biology and clinical behaviour of the tumors that arise .
this has not been widely evaluated in an african population where the incidence of breast cancer is rapidly increasing and where disparities exist between breast cancers among caucasian women in industrialized and african women in developing countries .
some studies indeed suggest that risk factors may vary by molecular subtypes therefore suggesting different etiologic pathways [ 810 ] . among the disparities based on tumor biology
the purpose of this study therefore was to establish the differences between er status and the traditionally known risk factors for breast cancer .
the country is 241,040 km and currently has a population of 32,709,865 people . with a total fertility rate of 6.7 births per woman -
the second highest in the world - uganda is due to double its population ( starting with 2,006 numbers ) by 2037 .
the capital , kampala , is a city of 1.4 million people located in the south - central region of the country . despite achieving 5.8% gross domestic product ( gdp ) growth rate in 2010 ,
almost one - third of the country still lives in poverty ( defined as living on less than us $ 1.25/day ) .
a total of 85% of the population live in rural areas , and most of them work in the agriculture sector .
uganda ranks 143 among the 169 countries surveyed for the 2010 human development index [ 16 , 17 ] .
life expectancy has slowly been increasing to its current level of 53 years , although half of the populations are between the ages of 0 and 14 years .
the ugandan health system is developed with public and private providers ; most of the health care is free , and referral for cancer care is mostly to mulago national referral hospital .
two tertiary care institutions : mulago hospital , the makerere university teaching hospital and ugandan cancer institute .
women with a confirmed histological diagnosis of invasive breast cancer were recruited prospectively and consecutively during the period of 2011 to 2012 .
we used the quality assurance guidelines of the college of american pathologists ( cap ) .
the laboratory had control tissue which was proven positive for each of the antibodies ; a section of the positive control was used at every run of the day , and a negative control was also run .
the slides were paraffinized and rehydrated in xylene followed by graded alcohols then washed in tris - buffered saline .
the immunohistochemical ( ihc ) assays were performed using an immunostainer with antibodies and antigen unmasking .
the results were scored semiquantitatively using reiner 's four point scale based on intensity and percentage of ihc reaction , and her2 staining was evaluated according to manufacturer 's instructions .
antibodies used were er ( clone sp - i ) , asr pr ( clone y85 ) , asr , and her2/neu ( c - erbb-2 ) , clone cb-11 ) and were purchased from cell marquee corporation , rocklin , ca , usa .
proxies of these molecular subtypes were determined by immunohistochemical stains of oestrogen receptor ( er ) , progesterone receptor ( pr ) , and human epidermal growth factor receptor ( her ) -2/neu proteins .
together the three markers were used to define four tumor subtypes : luminal a ( er= or pr+ and her-2 neu ) , luminal b ( er+ or pr+ , her-2/neu+ ) , her2/neu+ ( er & pr ) , and triple negative ( er , pr and her2/neu ) .
er / pr scoring system staining of < 5% of tumor cell nuclei was considered negative . both border line and overtly positive stains
her2/neu was considered when no staining was observed or membrane staining was < 10% of the tumor cells .
haematoxylin and eosin ( h&e ) staining was performed first to confirm diagnosis of invasive breast cancer before immunoassaying .
all the histological slides were received by an experienced consultant pathologist and laboratory technicians , and the tumors were classified according to nottingham modification of the scorff bloom richardson criteria . based on histology
, tumors were classified into the following groups : invasive ductal carcinoma ( nos ) , lobular , medullary , papillary , and colloid .
demographic factors and selected variables ( reproductive factors ) were compared between er+/er using t - tests and chi square tests for categorical variables .
data were occasionally missing for some variables , and no further analyses were done for missing data .
the study was approved by the institutional review boards of makerere university and the uganda national council of science and technology ( uncst ) .
table 1 presents the tumor characteristics by er status ; we considered the age equal to or below 50 years and above 50 years .
there were no differences between the er and the er+ tumors , though there were more of > 50 year olds in the er group .
for the rural and urban residences , the frequencies were nearly the same for both groups , so was the stage at presentation .
generally put the stage i & ii were considered early disease and stage ii & iv late or advanced disease . for tumor grade ,
the differences were statistically significant ( p = 0.001 ) with more of the high grade ( iii ) tumors falling in the er category .
.
table 2 presents invasive breast cancer er subtypes and risk factors . over all there were no statistically significant differences between reproductive factors ( parity , afb , breast feeding , and contraceptive use ) and er+/ tumors and no differences between nonreproductive factors ( age , premenopausal / menopausal , setting : rural or urban , and bmi ) , and all p values were > 0.05 .
we set out to investigate the extent to which risk factors both reproductive and nonreproductive for breast cancer among indigenous ugandan women differ by er status .
we anticipated that there were differences based on risk factors , due to differences in aetiological or and tumor progression pathways , as previously suggested in other studies [ 1921 ] .
we indeed found some clinical differences in characteristics by er status , supporting the view of heterogeneity and perhaps differences in aetiological pathways but no significant differences were found by risk factors ( especially the reproductive ones ) . over all , er+ cancers are numerically predominant and consistent with other studies . even though the sample size was small
there is some indication that er+ tumors included a higher percentage of lobular tumors , similar to other studies ; however the frequency of poorly differentiated tumors was the same across the er groups unlike in other studies .
this scenario of high grade tumors is consistent though with the overall picture encountered in sub - saharan africa where most women present with late stage disease with mostly poorly differentiated tumors [ 2226 ] .
the lack of significant differences between er status and risk factors could be interpreted in two ways : first , that the environmental risk exposure for both er and er+ group is similar , being in a rural or urban setting did not make a difference .
reproductive factors such as contraceptive use , parity and breast feeding were not any different between the two er groups .
there were more women with higher parity 7 in the er group by a factor of two , and implications of this high parity and er status are unclear .
the literature comparing risk factors by er status reveal inconsistent results , where some demonstrate differences [ 19 , 20 ] while others do not [ 21 , 27 ] .
the second explanation could be derived from the knowledge of the origins of er receptor status that all early premalignant breast lesions are strongly er positive .
mitogen activated protein kinase ( mapk ) activation and methylation of er promotor reversibly transforms er positive to er negative breast cancers .
the idea is that er expression is suppressed during tumor progression ; therefore the er status may depend significantly on the stage at diagnosis .
the mechanisms for loss of expression include reversible hypermethylation of the er promotor that downregulates er expression in invasive breast cancer tumors .
the marked difference in characteristics between the er+ and er groups lie in tumor grade , and the er group had more high grade tumor compared to er+ group by a factor of more than two .
this is consistent with previous literature and the knowledge that er tumors are more aggressive and carry a poorer prognosis .
what is also clear from these data is that there were more er than er+ tumors , a state of overrepresentation of er negative tumors or underrepresentation of er+ tumors .
the reasons why the er group would have more high grade tumors may be a factor of underlying genetic influence ( somatic or germline mutations ) .
what we may require moving forward is genetic exploration studies of er tumors in the african environment .
the alternative view is that er+ tumors are underrepresented , and this could be because of the population age structure influencing a predominantly premenopausal breast cancer presentation .
other studies suggest that the activation of the mapk receptors at the cell surface can downregulate er expression in a reversible manner , raising hope that reversing er expression may improve therapeutic response of these tumors .
it is possible that this picture is influenced by relatively small samples , though the findings are consistent with many others in the sub - saharan africa ; perhaps a multicenter larger study across countries is warranted .
ihc is performed in more clinical laboratories , it is less expensive and less labour intensive than fish which requires fluorescence microscopes rather than the light microscope used for routine microscopic evaluation by pathologists .
the omission of fish could have led to an underestimation of her2 + tumors though it would not affect the overall er proportions .
er negative tumors had significantly higher grade tumors , and no difference in risk factors by er status was found contrary to what some past studies suggest . given the disparities seen between african and caucasian women , a further wider inquiry is warranted for sub - saharan africa . | introduction .
breast cancer is the commonest cancer among women globally . in uganda
, it is on the rise , projected at a 4.5% annual asr increase ( age standardized incidence rate ) .
the reasons for this steep increase are not fully established . in the recent past ,
gene profiling in tumor tissues suggests that breast cancers are divided into subtypes dependent on the presence or absence of oestrogen receptor , progesterone , and human epidermal growth factor receptor 2 ( her 2 ) .
these subtypes do have distinctive clinical outcomes and perhaps risk factors from past studies .
there is paucity of data on hormonal receptor status and the traditionally known risk factors in sub - saharan africa .
the purpose of this study therefore was to establish the differences between er status and the traditionally known risk factors for breast cancer in uganda .
methods . an observational analytical hospital , based study , carried out at makerere university , college of health sciences .
formalin fixed and paraffin imbedded sections were prepared for haemotoxylin and eosin ( h&e ) stains and immunohistochemistry ( ihc ) .
ethical approval was obtained .
results . a total of 113 women were recruited .
mean age was 45 years ( sd14 ) .
there were no significant differences in selected risk factors ( setting , age , contraceptive use , parity , breast feeding , or menarche ) by er status although er negative tumors had significantly higher grade tumors ( by a factor of two ) compared to er positive tumors .
conclusion .
there were no significant differences among risk factors by er status contrary to what several other studies suggest . the manifestation of breast cancer in africa warrants further extensive inquiry . |
lateral dislocation of the patella is commonly encountered while intraarticular dislocations are rare and involve rotation of the patella around its horizontal or vertical axis.1 the vertical type occurs when the articular surface of the patella faces medially or laterally , indicating a rotation about a vertical axis , with lateral shifting being more common of two types.2 dislocations associated with rotational elements can be difficult to reduce by closed methods and can have associated osteochondral injuries.2 the rarity of these dislocations is evident by the lack of their description in text.3 of the few vertical dislocations reported in literature , the reported mechanism is of a severe driving force to the medial or lateral aspect of the patella with the knee extended , resulting in disruption of the retinacula .
the present report describes the characteristics of a neglected patellar dislocation with rotation on its vertical axis , its possible mechanism of injury , and management .
a 6 year - old male child , sustained injury to right knee and foot while stepping down from bus and bus suddenly picked speed , injuring his right knee and foot .
he fell down from the bus and subsequently his foot was run over by the bus .
he presented to the emergency in a primary health center with a fracture calcaneus and a grossly swollen and bruised knee .
a plaster of paris boot cast was given for fracture calcaneus and the knee injury was managed as a soft tissue injury .
the patient continued to have persistent pain in right knee swelling and difficulty in walking , hence reported to us 2 months after initial injury .
physical examination of the right knee revealed tenting of skin anterolaterally , notable lateral fullness , and the patella was slightly tender , immobile , and locked .
the knee could not be flexed because of pain and tautness of soft tissue around the patella , though overlying skin was mobile and normal .
radiological examination revealed a laterally wedged patella in ap view with no patellar shadow in lateral view [ figure 1 ] .
( a ) preoperative anteroposterior x ray of the knee shows laterally wedged patella ( arrow ) ; ( b ) lateral x - ray showing absence of patellar shadow since closed reduction was not feasible hence , under general anesthesia , an open reduction was planned . through midline incision ,
the knee was exposed and patella was found to be wedged lateral to lateral femoral condyle .
there was a continuous sheet of fibrous tissue from patella to medial extensor retinaculum , giving no indication of rotational element of patella . suspecting a clockwise rotation on its vertical axis due to its lack of mobility , an initial small incision was made on the surface of patella , which confirmed that the articular surface of patella was facing antero - laterally [ figure 2 ] .
as a result , the fibrous tissue medial to patella was incised by a longitudinal incision and patella derotated in an anticlockwise direction to bring it to its normal position .
the lateral extensor retinaculum being lax stretched over the articular surface of patella and did not require any release . instead
the knee was placed through a full range of motion and normal patellar tracking was noted .
( a ) intraoperative picture showing dislocated patella with articular surface facing anterolaterally ; ( b ) lateral retinaculam covering the articular surface of dislocated patella postoperatively , the knee was kept in full extension with a knee immobilizer , but was allowed full weight bearing and perform isometric quadriceps strengthening exercises .
he had no symptoms of patellar instability , normal muscle strength , and exhibited full range of motion [ figures 3 and 4 ] .
( a ) postoperative lateral x - ray showing normally located patella ; ( b ) postoperative anteroposterior x - ray showing absence of laterally wedged patellar shadow clinical photograph showing ( a ) full extension ; ( b ) full flexion
dislocation of the patella with vertical axis rotation was originally described by cooper in 1844.4 this is an exceedingly rare injury , having been reported only a few times since then [ table 1 ] .
ofluoglu et al . have recommended classification into two main groups depending on the location of the patella in the patello femoral joint.11 in intraarticular dislocations , the patella remains in its anatomical position and is only rotated around its vertical or horizontal axis . in extraarticular dislocations , the patella is displaced outside the patello femoral joint .
according to this classification , the present dislocation can be classified as an extraarticular lateral dislocation with rotation on the vertical axis .
although the exact mechanism of injury in vertical dislocation of patella is not clear , most often , these injuries are associated with sporting activities and occur as a result of direct blow to the medial aspect of patella with knee in near extension or a twisting injury to lower limb with forced internal rotation of the femur on externally rotated planted tibia while the knee is flexed .
the patella rotates on its vertical axis and gets wedged within intercondylar groove or outer surface of lateral femoral condyle.12 laxity of the ligaments allowing for greater mobility of the patella was thought to be a predisposing factor in adolescent patients.13 colville suggested that a valgus strain on the knee could cause the patella to rotate on its vertical axis and the continued pull of the quadriceps would then hold the patella on its edge.1014 the diagnosis is easily made on physical examination .
some authors have reported success with closed reduction under conscious sedation.15 in a recent review , open reduction was recommended to reduce the risk of chondral damage by repeated attempts at manipulation and to repair or remove any osteochondral fractures.13 the malrotated patella results in increased tension within the extensor mechanism , which in turn locks the patella between the femoral condyles , making manipulation difficult .
the present case involves a lateral dislocation of the patella in a male adolescent , which occurred with a direct blow .
the mechanism of injury was a laterally directed blow to the medial aspect of the knee with a twisting force due to fall .
the patella was dislocated with more than 90 rotation about its vertical axis , wedged lateral to the lateral femoral condyle with articular surface facing anterolaterally .
the increased tension generated by the rotation of the lateral extensor retinaculum kept the patella locked in the lateral gutter even with the knee in full extension .
in such a situation , the closed manipulation is unlikely to succeed , more so when the dislocation has become old and neglected .
have advocated multiple attempts at closed reduction in their case report , but in a fresh case10 one should suspect a rotatory component and proceed to open reduction without further attempts at closed reduction , which will only serve to increase any chondral damage to the patella or the condyle . in a fresh case , gap in the medial retinaculam
gives an indication of the location of damage and clue to rotational element of the dislocation .
this is not seen in a neglected case due to healing of the damaged medial retinaculam by fibrosis , making it look like a conventional lateral dislocation .
most of the patellar dislocations require lateral release procedures for maintaining stability due to tight lateral structures . however , an initial lateral release , with medial retinaculum already disrupted , is likely to compromise the blood supply of patella on both sides besides adding to the instability .
rotational element has to be recognized before incising the retinaculum ; otherwise , one is likely to incise the lateral retinaculum as well , further jeopardizing the blood supply to patella . to conclude the vertical patellar dislocation is uncommon and | patellar dislocations occurring about the vertical and horizontal axis are rare and irreducible .
the neglected patellar dislocation is still rarer .
we describe the clinical presentation and management of a case of neglected vertical patellar dislocation in a 6 year - old boy who sustained an external rotational strain with a laterally directed force to his knee . initially the diagnosis was missed and 2 months later open reduction was done .
the increased tension generated by the rotation of the lateral extensor retinaculum kept the patella locked in the lateral gutter even with the knee in full extension .
traumatic patellar dislocation with rotation around a vertical axis has been described earlier , but no such neglected case has been reported to the best of our knowledge . |
in the past two decades , carbon nanostructures and materials such as fullerenes , carbon nanotubes ( cnts ) , and graphene have been used in many practical devices .
furthermore , these materials possess the potential for further applications in stateoftheart materials science and nanoscaled electronics.1 , 2 , 3 , 4 , 5 this can be attributed to the abundant hybridization states ( sp , sp , sp ) of carbon , which affords the capacity to form complicated networks .
this is a fundamental principle of organic chemistry and is also the basis for the existence of life . in recent years ,
graphene and its derivatives have elicited considerable attention due to their unique structures and properties.6 , 7 , 8 , 9 , 10 moreover , carbon chains composed of cc units are predicted to exhibit highly beneficial mechanical , chargetransport , electronic , and nonlinear optical properties.11 , 12 , 13 , 14 , 15 , 16 long atomic carbon chains have also been observed either inside multiwalled carbon nanotubes or as a linker between two nanoscaled graphene fragments.17 , 18 , 19 , 20 , 21 , 22 stateoftheart organic synthesis has also allowed the expansion of an original molecular system through the insertion of cc units into the chemical bonds of a molecule.23 these structures are defined as the carbomers of the original molecules.23 using this strategy , various fascinating molecules such as carbomerized benzene and carbomerized cubane24 , 25 , 26 , 27 , 28 have been synthesized . in 2010 , li et al .
fabricated largearea ordered graphdiyne films in highyield for the first time.29 structurally , graphdiyne can be considered the structure of onethird of the cc bonds in graphene inserted with two cc units .
this newly obtained species of carbon networks with delocalized electron systems are of particular interest owing to their electronic , optical , and geometric characteristics.30 , 31 , 32 , 33 , 34 the highly conjugated , carbonrich graphdiyne and its analogues featuring tunable structural and optoelectronic properties have also been recognized as promising candidates for use in nextgeneration electronic and optoelectronic devices.35 , 36 , 37 , 38 , 39 graphdiyne nanowalls , as well as onedimensional ( 1 d ) nanoribbons and nanotubes derived from twodimensional ( 2 d ) graphdiyne have also been obtained experimentally.40 , 41 , 42
most recently the 1 d extended graphdiyne nanowire shown in scheme 1 was reported by cirera et al . using surfaceassisted covalent synthesis,43 which is the basis of this work .
conjugated chains with structures similar to that of the extended graphdiyne wire , functionalized with chemical substituents , were also obtained.44 , 45 1 d materials have attracted substantial attention in recent years due to their unique properties that differ markedly from those of 2 d and threedimensional ( 3 d ) structures.46 , 47 furthermore , 1 d nanostructures are essential because they are the building blocks of various 2 d slabs and 3 d solidstate structures .
therefore , such extended graphdiyne nanowires would be new and interesting carbon materials that are widely anticipated for future applications .
the synthesized 1 d extended graphdiyne nanowire . using firstprinciple calculations , the 1 d nanostructure of graphdiyne was shown to be a 1 d semiconductor with a simple band structure and a desirably small band gap.43 nevertheless , modulation of the electronic band gap is essential for future applications of semiconductors .
this is because the application of such new organic polymerbased semiconducting optoelectronics requires a much more thorough understanding of the nature of the electronic structure and chargetransport properties.48 , 49 various physical or chemical methods have been suggested50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 in order to realize this goal . for instance
, the electronic structures of cnts can be readily manipulated under electric fields . in some cases ,
a semiconductor / metal transition can even be achieved.50 , 51 the effects of substituent groups on the band gap and charge carrier mobility of graphene nanoribbons ( gnrs ) has been explored by wang and ge.52 previous studies have acknowledged that the addition of cc units into the chemical bonds not only expands the skeleton , but also opens a band gap of original graphene.53 , 54 therefore , carbomerization would be a good strategy for modifying the functional properties of lowdimensional nanostructures .
the aim of this work is to determine how an electric field , chemical functionalization at the benzene edges , and carbomerization affect the electronic properties of the recently obtained 1 d extended graphdiyne nanowires .
herein we report theoretical studies of the 1 d extended graphdiyne nanowires using a firstprinciple selfconsistent field crystal orbital ( scfco ) method under the framework of density functional theory ( dft ) .
the electronic structures of the extended graphdiyne nanowires are calculated under electric fields with varying strengths in the range of 0.253.0 v ( the step size is 0.25 v ) .
the field is applied in a direction perpendicular to the wire axis and parallel to the plane ( i.e. , along the yaxis direction of the coordinate system ; scheme 1 ) of the extended graphdiyne nanowire .
we first examined the case without an applied external field . based on the band structure shown in figure 1 a
, it is clear that the extended graphdiyne nanowire has a direct band gap ( e
g ) between the top of the highest occupied band ( hob ) and the bottom of the lowest unoccupied band ( lub ) .
therefore , the nanowire can be classified as a semiconductor with a band gap of 1.762 ev using the pbe method ( 2.605 ev with hse06 ) .
this is similar to the 1.60 ev value obtained from previous dft calculations by cirera et al.43 we then examined the results after the application of an external electric field .
it was observed ( figure 1 ) that the energy band levels are changed somewhat .
however , the shapes of the bands are well preserved when an electric field is applied .
a band gap is always present whenever an external field with varying strengths is applied .
these results suggest that the applied electric field can not alter the semiconducting property of the 1 d nanowire .
moreover , the change of the band gaps from 1.762 to 2.000 ev with pbe ( or 2.605 to 2.839 ev with hse06 , see table s1 , supporting information ) is not substantial .
the band gaps also exhibit an increasing trend when the field strength is enhanced to 3.0 v .
however , according to previous dft calculations , the band gaps of cnts , gnrs , and graphdiyne strips can decrease and sometimes even vanish with an increasing field.50 , 51 , 60 , 61 thus , the specific response of a 1 d extended graphdiyne wire to an electric field is antithetical to the behavioral trends observed in cnts , gnrs , and graphdiyne nanoscaled strips .
because the band gaps become even larger with the application of an electric field , we may expect that the applied electrical field would not cause a semiconductor / metal transition in the 1 d extended graphdiyne nanowires .
this fact is essential to the construction of electronic nanodevices , as the semiconducting property of the 1 d extended graphdiyne nanowire can be easily maintained even with an electric field .
electronic band structures of extended graphdiyne nanowire under a ) zero field and b ) external fields with various strengths ( in v ) .
the band gaps are 1.762 , 1.769 , 1.788 , 1.822 , 1.868 , 1.928 , and 2.000 ev under the external fields of 0.0 , 0.5 , 1.0 , 1.5 , 2.0 , 2.5 and 3.0 v , respectively .
the band structures in figure 1 show that the energy levels at the bottoms of the lubs are very close , with a range of 1.4681.535 ev under varying field strengths .
the energy levels for the tops of the hobs range from 3.578 to 4.001 ev , which means that there is a wider range for the hob energy levels than the lub energy values ( 0.423 vs. 0.067 ev ) .
thus , the band gap differences that are induced by the electric field are mainly attributed to variations in the hob in the extended graphdiyne nanowire . to gain more insight into the band gap variations under electric fields , the highest occupied crystal orbital ( hoco ) and the lowest unoccupied crystal orbital ( luco ) of the 1 d extended graphdiyne nanowire
the crystal orbital delocalizes in a longitudinal direction within the carbon chain fragment for both the ho and lu orbitals . however , the situation is rather different for the phenylene fragment . the crystal orbital spreads vertically in a longitudinal direction in the hoco and along the longitudinal direction in the luco .
the hoco of the extended graphdiyne nanowire is more drastically influenced by an applied field because it is oriented parallel to the field .
this influence is less significant in the luco because the crystal orbital distribution is always vertical to the applied electric field .
this explains why the energy levels of the hob for extended graphdiyne nanowires are more sensitive to electric fields than the energy level of the lub .
hoco ( upper ) and luco ( lower ) of the 1 d extended graphdiyne nanowire under a ) zero field and b ) electric field of 1.0 v . because the prepared graphdiyne nanowire can be treated as a junction of the polyphenylene and atomic carbon chain , calculations of the 1 d polyphenylene and carbon chain under an electric field were also performed using the same computational methods in order to compare the obtained values .
as shown in figure 3 , the band gaps of the isolated 1 d polyphenylene and the carbon chain with bond length alternation ( bla ) are 2.111 and 0.228 ev ( close to 0.34 ev by previous dftgga calculations62 ) , respectively , in the absence of an electric field . the application of an increasing electric field causes the band gaps of polyphenylene to also increase , which was similarly observed in the extended graphdiyne wire .
figure 3 also shows that the band gaps vary more rapidly for polyphenylene than 1 d extended graphdiyne wire .
relationship of band gap and field strength for the 1 d extended graphdiyne nanowire .
this implies that the band gaps of polyphenylene are more flexible if an electric field is applied .
however , the band gaps of the atomic carbon chain under the electric fields are nearly unchanged ( < 0.01 ev compared with the absence of an applied field ) .
this difference can be attributed to the distinct hoco and luco shown in figure 2 .
the hoco and luco are both oriented along the direction of the wire axis for the carbon chain , whereas the hoco and luco for polyphenylene are oriented in a vertical and parallel fashion , respectively .
studies of the extended graphdiyne wire show that the orbital distributions fall between the isolated polyphenylene and atomic carbon chain . therefore , the effects of band gap modulation with an electrical field for polyphenylene are greater than both the extended graphdiyne nanowires and the carbon chain .
to gain more quantitative information about the relationship between the band gaps and the field strengths , the band gaps e
g ( in ev ) of the extended graphdiyne wire under an applied electric field strength e ( in v ) are fit to the equation : e
g=0.024 e
+ 1.760 using the pbe method ( e
g=0.023 e
+ 2.600 using hse06 method ) with a correlation coefficient of r>0.99 . by using this equation , it is possible to accurately control the band gap of the 1 d extended graphdiyne nanowires by tuning the strength of the electric field .
scheme 2 shows eight models of the extended graphdiyne nanowires with chemically functionalized polyphenylene edges .
the functional groups in these models include three activating groups ( oh , nh2 , and ch3 ) , four deactivating groups ( f , cn , no2 , and cooh ) , and a combination of both activating ( ch3 ) and deactivating ( no2 ) groups . in this work , substitution of the h atoms occurs only at the r r positions ( scheme 2 ) .
this is because the atoms would be overly crowded and thus result in rippled edges52 if all of the 12 hydrogen atoms at the edges of the polyphenylene rings were substituted by functional groups .
the obtained electronic band structures with the various substituents are shown in figure 4 .
band structures of the 1 d extended graphdiyne nanowire with group substitutions as indicated at top .
figure 4 shows that the extended graphdiyne nanowires with either activating or deactivating functional groups are still semiconductors with direct band gaps .
this is quite similar to the trends observed in gnrs with varying functional groups.52 thus , the 1 d extended graphdiyne nanowire could maintain its semiconducting property even with various functional group substitutions .
table 1 shows that the obtained band gaps of the substituted extended graphdiyne nanowires have a range of 1.0231.708 ev using the pbe method ( 1.9612.374 ev using hse06 method ) .
these band gap values are all smaller than the value of 1.762 ev ( 2.605 ev with hse06 ) for pristine extended graphdiyne nanowire .
it is also found that there is a decline in the energy levels of both the hob and the lub for the deactivating groups ( f , cn , no2 , and cooh ) , and an increase in the energy levels for the activating groups ( oh , nh2 and ch3 ) .
[ a ] units : hob , lub , e
g , e
1v , and e
1c in ev ; c in ev ; m
h and m
h in m
e
;
h and
e in cm v s. because these extended nanowires are considered to be good candidates for nanoscaled electronics , an understanding of the charge carrier transport property would be advantageous .
we studied the charge carrier mobility of the extended graphdiyne nanowires with varying functional groups based on a theoretical model using a deformation potential ( dp ) theory and an effective mass approach.63 , 64 this model has been used for various carbon nanostructures such as fullerene chains , cnts , and gnrs.56 , 65 , 66 the charge carrier mobility of a 1 d crystal can be expressed as:(1)=em *
where m * is the effective mass of charge carriers , and can be obtained by fitting the band structures;(2)m*=22ek2 - 1
is the average scattering relaxation time of the charge carrier . with the dp approximation ,
of the 1 d system
is:(3)=2c2m*kbt1/2e12
where c is the stretching modulus of a 1 d crystal and can be obtained by:(4)c = l02el2l = l0
where l ( l
0 ) is the lattice constant ( equilibrium lattice constant ) .
e
1 ( including e
1c and e
1v ) are dp constants for charge carriers ( electron and hole ) and can be obtained by:(5)e1=l0l
detailed descriptions can be referenced from our previous studies.53 , 63 notably , only longitudinal acoustic ( la ) phonon scattering is considered in the models adopted here .
other scattering mechanisms such as optical phonon scattering , intersubband scattering and radialbreathing phonon scattering that can also limit the charge mobility , are not addressed in this work .
the la phonon scattering is the main scattering mechanism due to its high coupling strength even at room temperature according to previous studies.63 , 64 , 67 , 68 at low temperatures the optical phonon scattering is suppressed , and the electron scattering time by optical phonon remains almost unchanged with temperature due to the few excitation events.68
the calculated electron and hole mobilities for the extended nanowires at room temperature ( t=298 k ) are listed in table 1 .
the electron and hole mobilities (
e and
h ) of the fabricated extended graphdiyne nanowire were calculated to be 1.50010 and 3.54710 cm v s , respectively .
thus , the transport of electrons in the extended graphdiyne nanowire is more favorable than the transport of holes .
the m * value of the hole and the electron are very similar , although m
h * is slightly larger than m
e*. therefore , the substantially larger electron mobility is mainly due to a smaller electron dp constant .
the dp constants , e
1v and e
1c , are related to the bandedge shift induced by the scattering of the acoustic phonon at the hoco and luco , respectively . as mentioned above , both the hoco and luco delocalize in a longitudinal direction within the section of the carbon chain ( figure 2 ) . like the polyphenylene fragment
, the hoco spreads vertically in the longitudinal direction , but the luco is still oriented along the longitudinal direction . thus the scattering by acoustic phonon for the hoco is stronger than the luco , which leads to a larger e
1v value than the e
1c value for the extended wire .
therefore , the electron has a greater mobility than the hole for the extended graphdiyne nanowire .
it was found that the charge carrier mobilities of the extended graphdiyne nanowires with no2 and cooh groups exhibit rather distinctive properties relative to pristine and other chemically functionalized nanowires . although there is a decrease in the e
1v values in comparison with pristine wire , the hole mobility of these two structures is rather low and close to zero .
this can be attributed to the extremely large effective mass ( > 10 m
e ) for the hole carrier , which is attributed to their rather flat ho bands ( < 0.005 ev ) . in the band structures ,
it is clear that the original ho2 band ( figure 1 a ) shifts upward and becomes the ho band when the no2 and cooh groups are added to the extended graphdiyne nanowire .
figure 5 shows the hoco and luco of the 1 d nanowire with the no2 group .
it is observed that the hoco in no2substituted nanowire is actually derived from the ho2 orbital of the pristine nanowire .
furthermore , the hoco in the no2substituted nanowire exhibits clear localized characteristics , and is remarkably different from the delocalized features of the original hoco of pristine nanowire .
this fact explains the rather flat electronic energy band and the extremely large effective mass for the hole carrier of the no2substituted nanowire .
the electron mobilities are 5.1610 and 1.2310 cm v s for the no2 and cooh substituted models , respectively , which are one or two orders of magnitude smaller than the pristine wire . because the stretching modulus and the effective mass are similar to the extended graphdiyne nanowire , the decreased electron mobility is mainly due to the larger dp constants for the two structures .
the obtained dp constants for the electrons are 3.8 and 5.3fold larger than the original nanowire if the no2 and cooh groups are added .
a comparison of the luco of the extended graphdiyne nanowire before and after the no2 group is added is shown in figures 2 and 5 . aside from the orbital distributions along the longitudinal direction , new branches that are oriented vertically relative to the longitudinal direction are also present in the added substituents .
thus , the scattering through acoustic phonon for the luco is stronger in the no2substituted nanowire , which leads to a larger dp constant .
therefore , the electron mobility is always decreased with the addition of group substituents .
a ) hoco and b ) luco of the no2functionalized extended graphdiyne nanowire . in the extended graphdiyne nanowires with f , cn , oh , nh2 , and ch3 groups
, it is found that the hole mobility still exhibits the same order as the pristine nanowire .
this is because the stretching modulus , effective mass , and dp constants are all similar to those of the nanowire without any functional group substituents .
the electron mobility is decreased by one order of magnitude relative to pristine wire if these groups are added .
the reasoning for this observation is similar to our previously mentioned explanation of vertical orbital distributions in the luco for the substituted extended graphdiyne nanowires .
furthermore , the electron mobility is greater than the hole for each of the nanowires regardless of the presence of substituents on the nanowires .
the synthesized extended graphdiyne nanowire can be seen as a junction with cc and polyphenylene as building blocks .
thus , the carbomerized23 graphdiyne nanowires , cgnwn ( scheme 3 ) are built through an extension of the cc unit in the carbon chain portion .
n denotes the number of cc units between the two fragments of polyphenylene . in this work
, we decided to vary the number n from 2 to 20 in order to study the band gap and mobility of the extended graphdiyne nanowire upon carbomerization .
as shown in figure s2 ( supporting information ) , the obtained bond lengths of cgnwn with n=1820 for the long and short cc bonds in the center of the carbon chain fragment are 1.303 and 1.263 , respectively .
molecular dynamics ( md ) simulations were also performed to further investigate the kinetic stability of the nanowires using the densityfunctionalbased tightbinding ( dftb ) method.69 we performed three md simulations for each structure at 300 , 800 , and 1200 k for 3 ps under constanttemperature and constantvolume conditions with the time step size set at 1 fs .
the structures obtained after the md calculations and the potential energies with time evolution are presented in figure s3 ( supporting information ) .
we found that these 1 d nanowires exhibit a moderate structural deformation under dynamic evolution , which was particularly true for the structures with long chains at high temperature .
however , they remain very stable even at 1200 k after 3 ps of md simulation . according to the md simulations ,
figure 6 shows the calculated band structures of cgnwn with n=3 , 5 , 8 , 9 , 10 , 15 , and 20 .
based on the band structures , it is clear that these extended nanowires have a direct band gap .
therefore , it can be concluded that the 1 d carbomerized nanowires are all semiconductors with direct band gaps .
these results suggest that the semiconducting property of these 1 d wires is independent of the cc chain sizes . besides , the direct band gaps for the nanowires cgnwn with n=3 , 5 , 9 , 15 are all present at the x point in the first brillouin zone , whereas they are at the point for n=8 , 10 , and 20 according to the band structures shown in figure 6 .
this is a very interesting phenomenon , as for all the carbomerized nanowires tested , cgnwn with n being even or odd always presented band gaps at the or x points , respectively , in the first brillouin zone .
thus it seems that the location of band gaps of these carbomerized nanowires is determined only by the number of cc units .
electronic band structures of cgnwn with n=3 , 5 , 8 , 9 , 10 , 15 , and 20 as indicated at bottom .
the obtained band gaps of the carbomerized nanowires are shown in figure 7 , and are calculated to fall within the range of 0.6791.762 ev using the pbe method .
these flexible band gaps open up the possibility of modulating the electronic properties of these 1 d extended nanowires by tuning the sizes of the atomic carbon chains .
furthermore , it is observed that elongation of the carbon chain correlates to a decrease in the band gap , which means that a greater number of cc units would reduce the band gaps for the carbomerized nanowires .
the band gap of atomic carbon chain with bla is only 0.228 ev , which is significantly smaller than 2.111 ev for 1 d polyphenylene as mentioned above . because these 1 d extended nanowires are viewed as a junction with building blocks consisting of polyphenylene and carbon chain , this offers an explanation as to why the 1 d wire with a greater number of cc units exhibits a smaller band gap . to obtain quantitative information on the relationship of the band gap to the composition of the extended nanowires
, we plotted the e
g value of cgnwn with respect to n in figure 7 .
the band gaps of the 1 d wires are fitted to the equation e
g=0.537 + 1.542exp(n/8.549 ) , for pbe calculations and e
g=1.338 + 1.593exp(n/8.892 ) for hse06 calculations with a correlation coefficient of r>0.99 .
this suggests that the band gaps of the hybrid nanowires will be primarily determined by the number of cc units . with these equations ,
it is possible to accurately control the band gaps by tuning the number of cc units in the 1 d extended graphdiyne nanowires .
the electron and hole mobilities were also calculated to further study the charge carrier transport properties of the extended nanowires .
the calculated electron and hole mobilities for the extended nanowires at room temperature ( t=298 k ) are shown in figure 8 .
it is found that the electron and hole mobilities (
e and
h ) are within the range of 2.319101.50010 cm v s and 3.547102.70910 cm v s for the electrons and the holes , respectively .
the mobility of organic semiconductors is usually less than 100 cm v s,67 , 70 which is lower than the values obtained for the extended wires .
however , the mobility of charge carriers for the 1 d carbomerized nanowires does draw close to the charge carrier mobility values for semiconducting cnts and gnrs.56 , 65 , 66 thus , the 1 d carbomerized nanowires presented herein may be better candidates for highmobility materials than organic semiconductors
. moreover , the electrons still have a larger mobility than the holes for the nanowires with short carbon chains ( cgnwn with n7 ) .
in contrast , the electron and hole mobilities are quite similar in carbomerized graphdiyne nanowires with very long carbon chains ( cgnwn with n12 ) .
charge carrier mobility of cgnwn .
we observed that the hole mobility increases as the sizes of the carbon chains increase in the extended nanowires .
for instance , the hole mobility is 3.54710 cm v s in cgnw2 , but enlarges to 2.70910 cm v s in cgnw20 .
the opposite is observed for the electron mobilities , which are decreased as the carbon chains become longer . for comparison
the obtained electron and hole mobilities are 1.42010 and 1.53410 cm v s , respectively . therefore ,
the carbomerized graphdiyne nanowires with long carbon chains ( cgnwn with n5 ) exhibit a smaller mobility by one or two orders of magnitude for both the electrons and holes than the pure carbon chain with bla .
the obtained stretching modulus , the effective mass of charge carriers , and the dp constants for the 1 d carbomerized nanowires are listed in table s2 ( supporting information ) in order to provide more insight into the charge mobility of the 1 d nanowires .
we first examine the stretching modulus , which exhibits an increasing trend within a range of 9398 e , which falls between 90 e for polyphenylene and 118 e for the carbon chain . for the dp constants , the values of e
1v decrease , whereas the values of e
1c increase as the nanowires become longer . as a matter of fact ,
both the e
1v and e
1c values in cgnw20 are substantially close to the values in atomic carbon chain with bla . for the effective mass
, it is found that the m * value of both the hole and the electron decreases with elongation of the carbon chain .
the obtained m
h * and m
e * values for cgnw20 are 0.0785 and 0.0750 m
e
, respectively .
we are aware that the m * for the carriers in cgnw20 are still about twofold larger than the atomic carbon chain ( 0.0346 and 0.0327 m
e
for m
h * and m
e * ) . because the stretching modulus and dp constants of cgnw20 are similar to the atomic carbon chain , the decreased mobility of cgnw20 is most likely derived substantially from the heavy carriers .
the frontier orbital of the atomic carbon chain is calculated in order to explain the change in the effective carrier masses for the carbomerized graphdiyne nanowires . as shown in figure s4 ( supporting information ) , the hoco and luco of the pure carbon chain exhibit perfect conjugation because all of the carbon atoms are exactly identical . because the frontier orbital of the synthesized graphdiyne nanowire in figure 2 can be seen as a combination of polyphenylene and a carbon chain ,
figure s5 ( supporting information ) shows that the system is well maintained along the extended wire axis in the carbomerized graphdiyne nanowires .
additionally , the relative contribution to the frontier orbital from the atomic carbon chain greatly increases when the carbon chain is elongated .
this leads to the fact that the frontier orbital of cgnwn with a larger n tends to approach that of the pure carbon chain . as a result ,
the m * values for both the hole and electron for cgnwn also decrease and become closer to the pure carbon chain values as n is increased .
nevertheless , m * is still larger than the carbon chain due to the presence of orbital derived from the polyphenylene part , although the longest extended nanowire in the studies included 20 cc units .
the electronic structures of the extended graphdiyne nanowires are calculated under electric fields with varying strengths in the range of 0.253.0 v ( the step size is 0.25 v ) .
the field is applied in a direction perpendicular to the wire axis and parallel to the plane ( i.e. , along the yaxis direction of the coordinate system ; scheme 1 ) of the extended graphdiyne nanowire .
we first examined the case without an applied external field . based on the band structure shown in figure 1 a
, it is clear that the extended graphdiyne nanowire has a direct band gap ( e
g ) between the top of the highest occupied band ( hob ) and the bottom of the lowest unoccupied band ( lub ) .
therefore , the nanowire can be classified as a semiconductor with a band gap of 1.762 ev using the pbe method ( 2.605 ev with hse06 ) .
this is similar to the 1.60 ev value obtained from previous dft calculations by cirera et al.43 we then examined the results after the application of an external electric field .
it was observed ( figure 1 ) that the energy band levels are changed somewhat .
however , the shapes of the bands are well preserved when an electric field is applied .
a band gap is always present whenever an external field with varying strengths is applied .
these results suggest that the applied electric field can not alter the semiconducting property of the 1 d nanowire .
moreover , the change of the band gaps from 1.762 to 2.000 ev with pbe ( or 2.605 to 2.839 ev with hse06 , see table s1 , supporting information ) is not substantial .
the band gaps also exhibit an increasing trend when the field strength is enhanced to 3.0 v .
however , according to previous dft calculations , the band gaps of cnts , gnrs , and graphdiyne strips can decrease and sometimes even vanish with an increasing field.50 , 51 , 60 , 61 thus , the specific response of a 1 d extended graphdiyne wire to an electric field is antithetical to the behavioral trends observed in cnts , gnrs , and graphdiyne nanoscaled strips .
because the band gaps become even larger with the application of an electric field , we may expect that the applied electrical field would not cause a semiconductor / metal transition in the 1 d extended graphdiyne nanowires .
this fact is essential to the construction of electronic nanodevices , as the semiconducting property of the 1 d extended graphdiyne nanowire can be easily maintained even with an electric field .
electronic band structures of extended graphdiyne nanowire under a ) zero field and b ) external fields with various strengths ( in v ) .
the band gaps are 1.762 , 1.769 , 1.788 , 1.822 , 1.868 , 1.928 , and 2.000 ev under the external fields of 0.0 , 0.5 , 1.0 , 1.5 , 2.0 , 2.5 and 3.0 v , respectively .
the band structures in figure 1 show that the energy levels at the bottoms of the lubs are very close , with a range of 1.4681.535 ev under varying field strengths .
the energy levels for the tops of the hobs range from 3.578 to 4.001 ev , which means that there is a wider range for the hob energy levels than the lub energy values ( 0.423 vs. 0.067 ev ) .
thus , the band gap differences that are induced by the electric field are mainly attributed to variations in the hob in the extended graphdiyne nanowire . to gain more insight into the band gap variations under electric fields , the highest occupied crystal orbital ( hoco ) and the lowest unoccupied crystal orbital ( luco ) of the 1 d extended graphdiyne nanowire
the crystal orbital delocalizes in a longitudinal direction within the carbon chain fragment for both the ho and lu orbitals . however , the situation is rather different for the phenylene fragment . the crystal orbital spreads vertically in a longitudinal direction in the hoco and along the longitudinal direction in the luco .
the hoco of the extended graphdiyne nanowire is more drastically influenced by an applied field because it is oriented parallel to the field .
this influence is less significant in the luco because the crystal orbital distribution is always vertical to the applied electric field .
this explains why the energy levels of the hob for extended graphdiyne nanowires are more sensitive to electric fields than the energy level of the lub .
hoco ( upper ) and luco ( lower ) of the 1 d extended graphdiyne nanowire under a ) zero field and b ) electric field of 1.0 v . because the prepared graphdiyne nanowire can be treated as a junction of the polyphenylene and atomic carbon chain , calculations of the 1 d polyphenylene and carbon chain under an electric field were also performed using the same computational methods in order to compare the obtained values .
as shown in figure 3 , the band gaps of the isolated 1 d polyphenylene and the carbon chain with bond length alternation ( bla ) are 2.111 and 0.228 ev ( close to 0.34 ev by previous dftgga calculations62 ) , respectively , in the absence of an electric field . the application of an increasing electric field causes the band gaps of polyphenylene to also increase , which was similarly observed in the extended graphdiyne wire .
figure 3 also shows that the band gaps vary more rapidly for polyphenylene than 1 d extended graphdiyne wire .
relationship of band gap and field strength for the 1 d extended graphdiyne nanowire .
this implies that the band gaps of polyphenylene are more flexible if an electric field is applied .
however , the band gaps of the atomic carbon chain under the electric fields are nearly unchanged ( < 0.01 ev compared with the absence of an applied field ) .
this difference can be attributed to the distinct hoco and luco shown in figure 2 .
the hoco and luco are both oriented along the direction of the wire axis for the carbon chain , whereas the hoco and luco for polyphenylene are oriented in a vertical and parallel fashion , respectively .
studies of the extended graphdiyne wire show that the orbital distributions fall between the isolated polyphenylene and atomic carbon chain . therefore , the effects of band gap modulation with an electrical field for polyphenylene are greater than both the extended graphdiyne nanowires and the carbon chain .
to gain more quantitative information about the relationship between the band gaps and the field strengths , the band gaps e
g ( in ev ) of the extended graphdiyne wire under an applied electric field strength e ( in v ) are fit to the equation : e
g=0.024 e
+ 1.760 using the pbe method ( e
g=0.023 e
+ 2.600 using hse06 method ) with a correlation coefficient of r>0.99 . by using this equation , it is possible to accurately control the band gap of the 1 d extended graphdiyne nanowires by tuning the strength of the electric field .
scheme 2 shows eight models of the extended graphdiyne nanowires with chemically functionalized polyphenylene edges .
the functional groups in these models include three activating groups ( oh , nh2 , and ch3 ) , four deactivating groups ( f , cn , no2 , and cooh ) , and a combination of both activating ( ch3 ) and deactivating ( no2 ) groups . in this work ,
substitution of the h atoms occurs only at the r r positions ( scheme 2 ) .
this is because the atoms would be overly crowded and thus result in rippled edges52 if all of the 12 hydrogen atoms at the edges of the polyphenylene rings were substituted by functional groups .
the obtained electronic band structures with the various substituents are shown in figure 4 .
band structures of the 1 d extended graphdiyne nanowire with group substitutions as indicated at top .
figure 4 shows that the extended graphdiyne nanowires with either activating or deactivating functional groups are still semiconductors with direct band gaps .
this is quite similar to the trends observed in gnrs with varying functional groups.52 thus , the 1 d extended graphdiyne nanowire could maintain its semiconducting property even with various functional group substitutions .
table 1 shows that the obtained band gaps of the substituted extended graphdiyne nanowires have a range of 1.0231.708 ev using the pbe method ( 1.9612.374 ev using hse06 method ) .
these band gap values are all smaller than the value of 1.762 ev ( 2.605 ev with hse06 ) for pristine extended graphdiyne nanowire . it is also found that there is a decline in the energy levels of both the hob and the lub for the deactivating groups ( f , cn , no2 , and cooh ) , and an increase in the energy levels for the activating groups ( oh , nh2 and ch3 ) .
electronic property and carrier mobility of the extended graphdiyne nanowires with group substitutions .
[ a ] units : hob , lub , e
g , e
1v , and e
1c in ev ; c in ev ; m
h and m
h in m
e
;
h and
e in cm v s. because these extended nanowires are considered to be good candidates for nanoscaled electronics , an understanding of the charge carrier transport property would be advantageous .
we studied the charge carrier mobility of the extended graphdiyne nanowires with varying functional groups based on a theoretical model using a deformation potential ( dp ) theory and an effective mass approach.63 , 64 this model has been used for various carbon nanostructures such as fullerene chains , cnts , and gnrs.56 , 65 , 66 the charge carrier mobility of a 1 d crystal can be expressed as:(1)=em *
where m * is the effective mass of charge carriers , and can be obtained by fitting the band structures;(2)m*=22ek2 - 1
is the average scattering relaxation time of the charge carrier . with the dp approximation ,
of the 1 d system
is:(3)=2c2m*kbt1/2e12
where c is the stretching modulus of a 1 d crystal and can be obtained by:(4)c = l02el2l = l0
where l ( l
0 ) is the lattice constant ( equilibrium lattice constant ) .
e
1 ( including e
1c and e
1v ) are dp constants for charge carriers ( electron and hole ) and can be obtained by:(5)e1=l0l
detailed descriptions can be referenced from our previous studies.53 , 63 notably , only longitudinal acoustic ( la ) phonon scattering is considered in the models adopted here .
other scattering mechanisms such as optical phonon scattering , intersubband scattering and radialbreathing phonon scattering that can also limit the charge mobility , are not addressed in this work .
the la phonon scattering is the main scattering mechanism due to its high coupling strength even at room temperature according to previous studies.63 , 64 , 67 , 68 at low temperatures the optical phonon scattering is suppressed , and the electron scattering time by optical phonon remains almost unchanged with temperature due to the few excitation events.68
the calculated electron and hole mobilities for the extended nanowires at room temperature ( t=298 k ) are listed in table 1 .
the electron and hole mobilities (
e and
h ) of the fabricated extended graphdiyne nanowire were calculated to be 1.50010 and 3.54710 cm v s , respectively .
thus , the transport of electrons in the extended graphdiyne nanowire is more favorable than the transport of holes .
the m * value of the hole and the electron are very similar , although m
h * is slightly larger than m
e*. therefore , the substantially larger electron mobility is mainly due to a smaller electron dp constant .
the dp constants , e
1v and e
1c , are related to the bandedge shift induced by the scattering of the acoustic phonon at the hoco and luco , respectively . as mentioned above , both the hoco and luco delocalize in a longitudinal direction within the section of the carbon chain ( figure 2 ) . like the polyphenylene fragment
, the hoco spreads vertically in the longitudinal direction , but the luco is still oriented along the longitudinal direction . thus the scattering by acoustic phonon for the hoco is stronger than the luco , which leads to a larger e
1v value than the e
1c value for the extended wire .
therefore , the electron has a greater mobility than the hole for the extended graphdiyne nanowire .
it was found that the charge carrier mobilities of the extended graphdiyne nanowires with no2 and cooh groups exhibit rather distinctive properties relative to pristine and other chemically functionalized nanowires .
although there is a decrease in the e
1v values in comparison with pristine wire , the hole mobility of these two structures is rather low and close to zero .
this can be attributed to the extremely large effective mass ( > 10 m
e ) for the hole carrier , which is attributed to their rather flat ho bands ( < 0.005 ev ) . in the band structures ,
it is clear that the original ho2 band ( figure 1 a ) shifts upward and becomes the ho band when the no2 and cooh groups are added to the extended graphdiyne nanowire .
figure 5 shows the hoco and luco of the 1 d nanowire with the no2 group .
it is observed that the hoco in no2substituted nanowire is actually derived from the ho2 orbital of the pristine nanowire .
furthermore , the hoco in the no2substituted nanowire exhibits clear localized characteristics , and is remarkably different from the delocalized features of the original hoco of pristine nanowire .
this fact explains the rather flat electronic energy band and the extremely large effective mass for the hole carrier of the no2substituted nanowire .
the electron mobilities are 5.1610 and 1.2310 cm v s for the no2 and cooh substituted models , respectively , which are one or two orders of magnitude smaller than the pristine wire . because the stretching modulus and the effective mass are similar to the extended graphdiyne nanowire , the decreased electron mobility is mainly due to the larger dp constants for the two structures .
the obtained dp constants for the electrons are 3.8 and 5.3fold larger than the original nanowire if the no2 and cooh groups are added .
a comparison of the luco of the extended graphdiyne nanowire before and after the no2 group is added is shown in figures 2 and 5 . aside from the orbital distributions along the longitudinal direction , new branches that are oriented vertically relative to the longitudinal direction are also present in the added substituents .
thus , the scattering through acoustic phonon for the luco is stronger in the no2substituted nanowire , which leads to a larger dp constant .
therefore , the electron mobility is always decreased with the addition of group substituents .
a ) hoco and b ) luco of the no2functionalized extended graphdiyne nanowire . in the extended graphdiyne nanowires with f , cn , oh , nh2 , and ch3 groups
, it is found that the hole mobility still exhibits the same order as the pristine nanowire .
this is because the stretching modulus , effective mass , and dp constants are all similar to those of the nanowire without any functional group substituents .
the electron mobility is decreased by one order of magnitude relative to pristine wire if these groups are added .
the reasoning for this observation is similar to our previously mentioned explanation of vertical orbital distributions in the luco for the substituted extended graphdiyne nanowires .
furthermore , the electron mobility is greater than the hole for each of the nanowires regardless of the presence of substituents on the nanowires .
the synthesized extended graphdiyne nanowire can be seen as a junction with cc and polyphenylene as building blocks .
thus , the carbomerized23 graphdiyne nanowires , cgnwn ( scheme 3 ) are built through an extension of the cc unit in the carbon chain portion .
n denotes the number of cc units between the two fragments of polyphenylene . in this work
, we decided to vary the number n from 2 to 20 in order to study the band gap and mobility of the extended graphdiyne nanowire upon carbomerization .
as shown in figure s2 ( supporting information ) , the obtained bond lengths of cgnwn with n=1820 for the long and short cc bonds in the center of the carbon chain fragment are 1.303 and 1.263 , respectively .
molecular dynamics ( md ) simulations were also performed to further investigate the kinetic stability of the nanowires using the densityfunctionalbased tightbinding ( dftb ) method.69 we performed three md simulations for each structure at 300 , 800 , and 1200 k for 3 ps under constanttemperature and constantvolume conditions with the time step size set at 1 fs .
the structures obtained after the md calculations and the potential energies with time evolution are presented in figure s3 ( supporting information ) .
we found that these 1 d nanowires exhibit a moderate structural deformation under dynamic evolution , which was particularly true for the structures with long chains at high temperature .
however , they remain very stable even at 1200 k after 3 ps of md simulation . according to the md simulations ,
figure 6 shows the calculated band structures of cgnwn with n=3 , 5 , 8 , 9 , 10 , 15 , and 20 .
based on the band structures , it is clear that these extended nanowires have a direct band gap .
therefore , it can be concluded that the 1 d carbomerized nanowires are all semiconductors with direct band gaps .
these results suggest that the semiconducting property of these 1 d wires is independent of the cc chain sizes . besides , the direct band gaps for the nanowires cgnwn with n=3 , 5 , 9 , 15 are all present at the x point in the first brillouin zone , whereas they are at the point for n=8 , 10 , and 20 according to the band structures shown in figure 6 .
this is a very interesting phenomenon , as for all the carbomerized nanowires tested , cgnwn with n being even or odd always presented band gaps at the or x points , respectively , in the first brillouin zone .
thus it seems that the location of band gaps of these carbomerized nanowires is determined only by the number of cc units .
electronic band structures of cgnwn with n=3 , 5 , 8 , 9 , 10 , 15 , and 20 as indicated at bottom .
the obtained band gaps of the carbomerized nanowires are shown in figure 7 , and are calculated to fall within the range of 0.6791.762 ev using the pbe method .
these flexible band gaps open up the possibility of modulating the electronic properties of these 1 d extended nanowires by tuning the sizes of the atomic carbon chains .
furthermore , it is observed that elongation of the carbon chain correlates to a decrease in the band gap , which means that a greater number of cc units would reduce the band gaps for the carbomerized nanowires .
the band gap of atomic carbon chain with bla is only 0.228 ev , which is significantly smaller than 2.111 ev for 1 d polyphenylene as mentioned above . because these 1 d extended nanowires are viewed as a junction with building blocks consisting of polyphenylene and carbon chain , this offers an explanation as to why the 1 d wire with a greater number of cc units exhibits a smaller band gap . to obtain quantitative information on the relationship of the band gap to the composition of the extended nanowires
, we plotted the e
g value of cgnwn with respect to n in figure 7 .
the band gaps of the 1 d wires are fitted to the equation e
g=0.537 + 1.542exp(n/8.549 ) , for pbe calculations and e
g=1.338 + 1.593exp(n/8.892 ) for hse06 calculations with a correlation coefficient of r>0.99 .
this suggests that the band gaps of the hybrid nanowires will be primarily determined by the number of cc units .
with these equations , it is possible to accurately control the band gaps by tuning the number of cc units in the 1 d extended graphdiyne nanowires .
the electron and hole mobilities were also calculated to further study the charge carrier transport properties of the extended nanowires .
the calculated electron and hole mobilities for the extended nanowires at room temperature ( t=298 k ) are shown in figure 8 .
it is found that the electron and hole mobilities (
e and
h ) are within the range of 2.319101.50010 cm v s and 3.547102.70910 cm v s for the electrons and the holes , respectively .
the mobility of organic semiconductors is usually less than 100 cm v s,67 , 70 which is lower than the values obtained for the extended wires .
however , the mobility of charge carriers for the 1 d carbomerized nanowires does draw close to the charge carrier mobility values for semiconducting cnts and gnrs.56 , 65 , 66 thus , the 1 d carbomerized nanowires presented herein may be better candidates for highmobility materials than organic semiconductors .
moreover , the electrons still have a larger mobility than the holes for the nanowires with short carbon chains ( cgnwn with n7 ) .
in contrast , the electron and hole mobilities are quite similar in carbomerized graphdiyne nanowires with very long carbon chains ( cgnwn with n12 ) .
we observed that the hole mobility increases as the sizes of the carbon chains increase in the extended nanowires .
for instance , the hole mobility is 3.54710 cm v s in cgnw2 , but enlarges to 2.70910 cm v s in cgnw20 .
the opposite is observed for the electron mobilities , which are decreased as the carbon chains become longer . for comparison ,
the obtained electron and hole mobilities are 1.42010 and 1.53410 cm v s , respectively .
therefore , the carbomerized graphdiyne nanowires with long carbon chains ( cgnwn with n5 ) exhibit a smaller mobility by one or two orders of magnitude for both the electrons and holes than the pure carbon chain with bla .
the obtained stretching modulus , the effective mass of charge carriers , and the dp constants for the 1 d carbomerized nanowires are listed in table s2 ( supporting information ) in order to provide more insight into the charge mobility of the 1 d nanowires .
we first examine the stretching modulus , which exhibits an increasing trend within a range of 9398 e , which falls between 90 e for polyphenylene and 118 e for the carbon chain . for the dp constants , the values of e
1v decrease , whereas the values of e
1c increase as the nanowires become longer . as a matter of fact ,
both the e
1v and e
1c values in cgnw20 are substantially close to the values in atomic carbon chain with bla .
for the effective mass , it is found that the m * value of both the hole and the electron decreases with elongation of the carbon chain .
the obtained m
h * and m
e * values for cgnw20 are 0.0785 and 0.0750 m
e
, respectively .
we are aware that the m * for the carriers in cgnw20 are still about twofold larger than the atomic carbon chain ( 0.0346 and 0.0327 m
e
for m
h * and m
e * ) . because the stretching modulus and dp constants of cgnw20 are similar to the atomic carbon chain , the decreased mobility of cgnw20 is most likely derived substantially from the heavy carriers .
the frontier orbital of the atomic carbon chain is calculated in order to explain the change in the effective carrier masses for the carbomerized graphdiyne nanowires . as shown in figure s4 ( supporting information ) , the hoco and luco of the pure carbon chain exhibit perfect conjugation because all of the carbon atoms are exactly identical . because the frontier orbital of the synthesized graphdiyne nanowire in figure 2 can be seen as a combination of polyphenylene and a carbon chain ,
figure s5 ( supporting information ) shows that the system is well maintained along the extended wire axis in the carbomerized graphdiyne nanowires .
additionally , the relative contribution to the frontier orbital from the atomic carbon chain greatly increases when the carbon chain is elongated .
this leads to the fact that the frontier orbital of cgnwn with a larger n tends to approach that of the pure carbon chain . as a result ,
the m * values for both the hole and electron for cgnwn also decrease and become closer to the pure carbon chain values as n is increased .
nevertheless , m * is still larger than the carbon chain due to the presence of orbital derived from the polyphenylene part , although the longest extended nanowire in the studies included 20 cc units .
on the basis of the firstprinciple dft calculations , modulation of the electronic properties of the 1 d extended graphdiyne nanowires was investigated by application of an electric field , chemical functionalization , and carbomerization .
crystal orbital analysis was also performed to further ascertain the modulation of electronic properties . when an electric field is applied in a direction perpendicular to the wire axis and parallel to the plane of the extended graphdiyne nanowire
, the nanowire still exhibits semiconducting properties , with band gaps ranging from 1.7622.000 ev . the relationship of the band gaps and the field strength was found to be e
g=0.024 e
+ 1.760 , which would be useful for accurately controlling the band gap of the 1 d extended graphdiyne nanowire based on the strength of the electric field .
the electronic properties of chemically functionalized 1 d extended graphdiyne nanowires were also studied with several incorporated groups ( oh , nh2 , ch3 , f , cn , no2 , and cooh ) at the polyphenylene edges .
the functionalized extended graphdiyne nanowires with either activating or deactivating groups are still semiconductors despite the fact that the band gaps are somewhat smaller than those of pristine extended graphdiyne nanowire .
the electron mobilities of the functionalized nanowires are one to two orders of magnitude smaller than those of pristine wire .
the hole mobility of the extended graphdiyne nanowires with no2 and cooh groups is rather low and close to zero because of their rather flat ho bands .
the carbomerized nanowires are built by elongation of the cc unit in the carbon chain fragments of the extended graphdiyne nanowires .
the band gap is fit to e
g=0.537 + 1.542exp(n/8.549 ) in terms of the number of cc units , which implies the possibility of tuning the band gaps by modifying the number of cc units in the extended graphdiyne nanowire .
the obtained electron and hole mobilities for the carbomerized nanowires have values in the ranges of 2.319101.50010 cm v s and 3.547102.70910 cm v s , respectively , which are clearly larger than the mobility values for organic semiconductors .
the modulation of band gap and carrier mobility of the extended graphdiyne nanowires influenced by several physical or chemical methods provides greater flexibility for energy band engineering as well as charge transport .
these interesting findings indicate that the extended graphdiyne nanowire and its analogues are promising candidates for future applications in novel nanoelectronics and molecular devices .
thus , we believe that these results regarding graphdiynebased materials will be helpful in moving these new fields forward .
the energies , band structures , and properties of the extended graphdiyne nanowires in this work were computed with full structural optimizations using the scfco method based on firstprinciple dft calculations with the crystal09 program.71 , 72 the exchangecorrelation functional is addressed using the method proposed by perdew , burke , and ernzerhof ( pbe).73 because we are aware that the pbe functional would underestimate the band gaps of semiconductors , the band structures of the extended graphdiyne nanowires were also calculated using the more sophisticated hybrid functional method , hse06 .
this method yields a more accurate approximation of the band gaps for carbonbased nanomaterials.33 a double plus polarization basis set 621g(d , p ) implemented in the program for solidstate calculations and an 80 kpoints sampling in the first brillouin zone were adopted in the scfco calculations .
the default values for the convergence criteria in crystal09 were used in our calculations . in the calculations , we assume that all of the extended graphdiyne nanowires exhibit a coplanar conformation , which is a common treatment of conjugated systems.43 , 74 , 75 it should be pointed out that the phenyl rings exhibit a flexible twist angle under different conditions.76 , 77 , 78 , 79 , 80 , 81 , 82 the twist angles are 3040 in the gas phase , whereas in the bulk phase they adopt a more planar conformation , with interring twist angles of 1020.76 , 77 , 78 moreover , several studies have indicated that some oligomers of the poly(paraphenylene ) adopt a planar configuration under high pressure , or on average with high librational amplitudes.81 , 82 actually , several conformations of the extended graphdiyne nanowire with twist angles in the range of 040 were also calculated with the same method for comparison . as shown in figure s1 ( supporting information ) , it was found that the obtained band structures of the conformations with different twist angles exhibit no qualitative difference , which agrees well with previous studies.43
as a service to our authors and readers , this journal provides supporting information supplied by the authors .
such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset .
technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors . | abstractgraphdiyne and derivatives with delocalized electron systems are of particular interest owing to their structural , electronic , and transport properties , which are important for potential applications in nextgeneration electronics . inspired by recently obtained extended graphdiyne nanowires , explorations of the modulation of the band gap and carrier mobility of this new species are still needed before application in device fabrication . to provide a deeper understanding of these issues , herein we present theoretical studies of onedimensional extended graphdiyne nanowires using firstprinciple calculations .
modulation of the electronic properties of the extended graphdiyne nanowire was investigated systemically by considering several chemical and physical factors , including electric field , chemical functionalization , and carbomerization .
the band gap was observed to increase upon application of an electric field parallel to the plane of the synthesized graphdiyne nanowire in a nonperiodic direction .
although chemical functionalization and carbomerization caused the band gaps to decrease , the semiconducting property of the nanowires was preserved .
band gap engineering of the extended graphdiyne nanowires was explored regarding the field strength and the number of cc units in the carbon chain fragments .
the charge carrier mobility of chemically functionalized and carbomerized extended graphdiyne nanowires was also calculated to provide a comparison with pristine nanowire .
moreover , crystal orbital analysis was performed in order to discern the electronic and charge transport properties of the extended graphdiyne nanowires modified by the aforementioned chemical and physical factors . |
the external environmental cues that set the body 's circadian clock are referred to as zeitgebers or time - givers by those who study circadian rhythms .
these external cues , in turn , entrain a cascade of neurohormonal events , such as diurnal patterns of cortisol and melatonin secretion , which are key components of circadian physiology .
the principal , and arguably most influential , zeitgeber is the rising and setting of the sun .
however , our modern society is no longer governed by the availability of natural light , as artificial light sources are readily available 24 hours a day . by extension , social factors such as the timing of meals , work schedules , the schedules of other family members , and even , to some extent , the timing of television programs can all have a substantial influence on an individual 's social rhythms and , in turn , on their circadian rhythms .
it is our belief that all of us are susceptible to the disruptive effects of changes in any of these important social time cues , and that these changes manifest themselves to varying degrees of temporary cognitive or somatic distress .
many of us have experienced the fatigue and malaise associated with the adjustment to a new time zone , but for most of us , these feelings generally dissipate quite rapidly .
however , we propose that those individuals who are susceptible to mood disorders find this sort of adjustment much more challenging .
in essence , they find themselves trapped in a sustained , disrupted cognitive and somatic state attributed to their perturbed circadian system . as an extension of this initial desynchronization , these individuals may then go on to experience fully syndromal manic or depressive episodes . indeed , since first articulating this social zeitgeber hypothesis of mood disorders , we established that life events with the capacity to alter daily routines are in fact associated with the onset of both depressive and manic episodes , with particular emphasis on the latter .
it is still unclear how , and to what extent , the body 's circadian system becomes disrupted in patients with mood disorders .
studies of seasonal affective disorder have pointed towards day length and/or exposure to light as possible factors in the development and treatment of the illness .
however , studies of nonseasonal depression have failed to corroborate the idea that light is the key synchronizing force in these individuals ' disorders .
given that the loss of social zeitgebers and/or the presence of life events with the capacity to disrupt daily routines are , in fact , implicated in the generation of new affective episodes in biologically susceptible individuals , then it would follow that effective treatment of bipolar disorder should center on principles of social rhythm stabilization , particularly as a preventative measure .
other empirically validated treatments for mood disorders already support this concept . cognitive therapy , with its focus on mastery and pleasure scheduling , and interpersonal psychotherapy , which emphasizes the resolution of role disputes and the easing of role transitions , both play substantial roles in establishing social routines , albeit via indirect means .
our research group has had a long - standing interest in the association between mood disorders and the circadian system . as a result
, we developed a therapeutic intervention for bipolar disorder that sought to prevent the recurrence of new affective episodes through indirect regulation of the endogenous circadian system .
the idea behind this approach , which we called interpersonal and social rhythm therapy ( ipsrt ) , was that if we could somehow increase the regularity of patients ' daily routines ( specifically , their often erratic sleep / wake cycles , meal times , and times of rest versus activity ) we could thereby help strengthen their otherwise vulnerable circadian systems .
ipsrt directly incorporates social rhythm theories into the framework of interpersonal psychotherapy , initially developed by klerman and colleagues for the treatment of unipolar depression .
specifically , ipsrt is geared toward stabilizing patients ' routines while simultaneously improving the quality of their interpersonal relationships and their performance of key social roles . through this approach
, ipsrt aims to improve patients ' current mood and level of functioning and to provide them with the skills necessary to shield them from new affective episodes .
although ipsrt was originally developed for individuals with bipolar i disorder , it now appears that ipsrt can be utilized in the treatment of both bipolar i and ii disorders . for patients with bipolar i disorder ,
acute affective symptoms are managed primarily through pharmacological means , with ipsrt used mainly as an adjunctive treatment to help regularize routines and improve social relations and role performance .
however , it now appears that ipsrt can be used as monotherapy for patients with bipolar ii disorder of moderate symptom severity ( swartz ha et al , unpublished data ) , or can be combined effectively with pharmacotherapy when such treatment is indicated .
regardless of the subtype of bipolar illness , we would argue that one key to managing mood symptoms lies in the regulation of social rhythms . for individuals with bipolar disorder
who are being treated with mood - stabilizing medications , recurrence vulnerability appears to occur via three main pathways : ( i ) nonadherence to medication ; ( ii ) presence of a stressful life event ; and ( iii ) disruptions in social rhythms .
ipsrt was designed with each of these potential vulnerability factors in mind , making it a targeted approach to treating this frequently recurring illness .
patients are provided with guidance and training on how to maintain a consistent medication schedule , an opportunity to discuss how they feel about the disorder itself and express their grief and/or anger over what we have frequently referred to as the lost healthy self , and a chance to come to grips with the often debilitating effect the illness has had on their lives . as a result , ipsrt often helps patients accept the life - long nature of their illness , reduces the denial commonly associated with the disorder , and thus facilitates medication adherence .
the behavioral component of ipsrt focuses on evaluating the degree to which the timing of a patient 's routines varies throughout any given week . to do this , we utilize a self - report charting instrument called the social rhythm metric ( srm ) , which allows the patient to keep track of when he or she goes to bed , gets out of bed , eats , goes to work , makes social contacts , etc .
table 1 shows an adapted version of the srm-5 . after reviewing the srm with the patient
, we then strive to help him or her make the timing of such routines more regular , ideally varying by no more than an hour .
this often needs to be done quite gradually , especially when specific routines vary by many hours over the course of a week .
when this is the case , we might choose to focus on just one routine , such a when the patient gets out of bed , attempting , by successive approximation , to approach an out of bed time that does not vary by more than an hour from day to day .
once reasonably regular routines are established , we review with the patient possible triggers to rhythm disruption that may surface in the near future ( le , house guests or vacations ) and work on strategies for maintaining the greatest amount of regularity despite the presence of these possible disruptions .
thus , ipsrt attempts not only to increase the stability of these rhythms , but also to increase the patient 's awareness of how easily these rhythms can be disrupted and how to manage in the face of such potential disruptions .
this behavioral approach to rhythm regularity is then interwoven with work on the four main problem areas targeted by klerman and colleagues interpersonal psychotherapy : unresolved grief , role transitions , role disputes , and interpersonal deficits . by addressing these interpersonal and social role issues with the patient ,
it is our hope that the number and severity of such stressors will decrease , thus making it easier to maintain the routine regularity stressed in the behavioral component of the treatment while at the same time enhancing self - esteem and social support .
indeed , there are several reasons why the reduction of interpersonal and social role stress is vital to achieving wellness in individuals with bipolar disorder .
first of all , stressful events have the capacity to impact the circadian system via increases in autonomic arousal that can , in turn , alter sleep - wake cycles , timing ( and amount ) of food consumption , and normal circadian patterns of release of other hormones .. second , regardless of the level of stress incurred , events of any size or severity can lead to significant changes in daily routines .
even a seemingly benign event , such as a child joining a sports team and needing to be at school an hour earlier for practice , can be difficult for someone struggling with bipolar disorder .
third , major life stressors such as moving house or getting a divorce can not only have a negative psychological impact on the individual , but may also disrupt social rhythms .
regardless of the patient 's clinical state at the beginning of treatment ( either in an acute episode or remission ) the first phase of treatment is always a focused history - taking . during this phase ,
the clinician seeks to establish the correct diagnosis and then to assess the linkage between acute episodes and interpersonal issues and social routines in the patient 's history , thus developing the foundation for treatment .
in addition to taking a detailed history , the clinician also takes the time to provide the patient and involved family members with education about the nature of bipolar mood disorder , being particularly careful to take into consideration what he or she may already know about the illness . also part of this initial phase of treatment
is an information - gathering process that we refer to as the interpersonal inventory ( ii ) . through this semistructured interview
, the therapist assesses the nature and quality of the patient 's current and past interpersonal relationships .
once these evaluations have been made , the clinician then proceeds to appraising the regularity of the patient 's social routines by using the srm .
the initial phase concludes with the selection of an interpersonal problem area ( unresolved grief , role transition , role disputes , or more pervasive interpersonal deficits ) , upon which the patient and the clinician both agree .
the combination of the history - taking and the ii should allow the clinician and patient to reach a consensus about which of these four problem areas is most closely linked to the onset of the most recent mood episode .
this focus then , becomes the initial jumping - off point for the interpersonal part of the therapy .
depending upon the severity and duration of the patient 's past psychiatric history , as well as the complexity of the patient 's current interpersonal relationships and level of insight into his or her own illness , this initial phase of treatment can last anywhere from three to five sessions .
once the first phase of treatment is completed , the clinician moves on to the second or intermediate phase of therapy .
the focus of this phase of the intervention involves helping the patient establish more regular dally social routines and resolve the interpersonal problem area specified in the initial phase of treatment . during this phase of ipsrt
however , depending upon the patient 's clinical status , more or less frequent sessions may be more appropriate .
in the two large trials of ipsrt conducted to date , this phase has typically required 10 to 12 sessions ; however , in a small open study of bipolar disorder complicated by full - criterion borderline personality disorder , a much longer intermediate phase - of the order of 9 to 10 months - was required to achieve mood stability .
the next phase of treatment , continuation or maintenance ipsrt , focuses on building up patients ' confidence in their capability to use the skills learned in the acute phase of treatment to maintain their current euthymic mood , level of functioning , and social rhythm regularity .
the objective is for the patient to be able to maintain regular social rhythms despite the probable occurrence of stressors such as job changes , vacations , and other unexpected life events .
additionally , the patient is encouraged to continue to improve the quality of his or her interpersonal relationships and keep the level of interpersonal distress at a minimum .
techniques that are commonly used for accomplishing these interpersonal goals include communication analysis , which allows the therapist and patient to identify problem areas in communication to help the patient interact more effectively with significant others ; role - play , which allows the patient a safe environment in which to practice expressing emotions and self - assertion ; and decision analysis , which helps patients to reflect on the potential risks and benefits of alternate choices and options with regard to a specific problem .
more detailed explanations of ipt techniques and strategies can be found in the manual for interpersonal psychotherapy .
treatment frequency generally decreases from weekly , to biweekly , and eventually to monthly sessions as the patient moves from acute to maintenance therapy . when termination of treatment is deemed appropriate ( or is otherwise necessitated by outside factors such as financial constraints or logistical challenges ) the clinician and patient will begin work on the final phase of treatment which focuses on impending termination
this can typically be accomplished within three to five monthly sessions . if it is not considered clinically appropriate to stop treatment completely , this final phase can also be utilized to further decrease the frequency of sessions to little more than occasional booster sessions when necessary .
ipsrt can also be successfully implemented simply as a short - term treatment , in which case the initial phase is more condensed and the work on the interpersonal problem areas must be more concentrated . despite the shortened overall duration of treatment in this case
, it is still advisable that treatment frequency be reduced near the end , so as to still allow for at least three to four bimonthly sessions to accomplish the necessary termination steps .
the following is a detailed example of how ipsrt is utilized in the treatment of a patient with bipolar i disorder .
anne is a 35-year - old separated woman who began ipsrt while in the throes of a particularly severe depression that had had its onset 4 months previously .
anne had been working as a waitress at a local restaurant , with a schedule that varied widely : she never had a consistent day off , and she was frequently scheduled for lunchtime shifts one day followed by evening shifts the next .
she had recently moved out the home she shared with her estranged husband and she was having great difficulty making ends meet , with the rental fees of her new apartment and the need to buy a car of her own .
as anne 's depression worsened , she found herself struggling with disrupted sleep and a lack of motivation which began to negatively impact her work .
she began showing up late for her shifts , and the fatigue stemming from her erratic sleep schedule caused her to make several mistakes with her customers .
soon she was in serious jeopardy of losing her job , the prospect of which sparked intense financial anxiety .
while anne had a rather protracted history of brief but severe manias , this appeared to be one of very few depressions anne had experienced in her life .
additionally , anne had married young ( at age 20 ) and had , for all intents and purposes , moved directly from her parents ' home into the home she shared with her husband .
she had never actually lived alone , and was finding her new circumstances as frightening as they were difficult .
anne 's therapist could see that there were several factors that would need to be addressed in order to help anne out of her depression .
first , anne 's schedule was far too erratic . in order for anne to be able to regulate her daily routines
second , it appeared to the clinician that anne 's primary interpersonal problem was the role transition from being a married woman to a single one and the challenges of self - sufficiency that came along with it .
the particularly stressful life event of her marital separation was also the impetus for her current disordered state of affairs , so work focused on ameliorating her interpersonal stress would be fundamental to achieving stable social rhythms . once anne 's therapist had completed the history - taking and interpersonal inventory , she and anne moved into the intermediate phase of treatment .
anne 's therapist first began problem - solving with anne about how to make her schedule more consistent at work .
after discussing the nature of anne 's relationship with her supervisor ( good until she had started missing work ) , her therapist suggested she talk with her supervisor about requesting set shifts on a weekly basis .
while anne knew that , because of the nature of the restaurant business , it would be nearly impossible for her to have the same days off each week , she agreed that her boss might be somewhat receptive to the idea of at least making her shifts take place during the same times each day , especially if it meant this would help her to be a more reliable employee . with anne 's history of mania ,
her therapist suggested that she avoid the late - night shifts if at all possible .
anne 's supervisor was in fact amenable to her requests , and upon successfully obtaining a more stable work schedule , anne and her therapist then went to work on regulating her sleep schedule . using the srm as a guide , anne and her therapist agreed on set times when anne would go to bed at night and get up in the morning , aiming to have these times vary by no more than an hour , even on her days off .
anne 's therapist offered her education on sleep hygiene , and explained how getting better sleep would not only help her mood , but would also make her less clumsy and forgetful at work , thereby alleviating some of her work - related stress and worry . while this behavioral work was being done to help regulate anne 's social rhythms , her therapist was simultaneously working with her on her role transition to being a single woman and dealing with the stress and loneliness she felt as a result of her marital separation .
her therapist stressed the importance of creating a solid support network to help her through this difficult time , encouraging anne to find ways to express her feelings about her current situation .
anne 's therapist knew from the ii that anne maintained a good relationship with her parents and had at least two female friends from high school with whom she remained close ; however , she rarely saw either her parents or these girlfriends because she felt too depressed to leave her apartment other than to drag herself to work .
she encouraged anne to visit her parents on one of her days off and to make some arrangements to see one of her friends on the other day .
anne noted a link between getting herself to her parents ' house and improvement in her mood .
she also found that after doing something with her friends , she felt better as well .
as anne continued to work on regularizing her daily routines and improving her satisfaction with her interpersonal relationships , she felt her depressive symptoms begin to dissipate .
anne 's therapist remained cognizant of anne 's history of mania , and kept a careful eye out for any signs that anne 's mood disorder was not actually remitting , but rather was cycling into an episode of mania .
she stressed that it was important for anne to not become too overstimulated ( especially considering the often hectic nature of her job ) , in the hopes of preventing a manic recurrence .
anne remained in the acute phase of treatment for approximately 22 weeks , and then moved into the maintenance phase of treatment .
after three biweekly sessions , anne and her therapist moved to monthly sessions where they focused on maintaining anne 's routine regularity and strove to stay ahead of any potential pitfalls to her progress , particularly the stress of impending divorce proceedings .
ipsrt has been supported empirically through two large studies involving the therapy in combination with pharmacotherapeutic interventions in the treatment of bipolar disorder .
the first of these studies involved 175 patients with bipolar i disorder who presented for treatment while in the midst of a depressive , manic , or mixed episode .
in this two - phase study , these individuals were randomly allocated to four acute and maintenance treatment sequences : acute and maintenance ipsrt ( ipsrt / ipsrt ) , acute and maintenance intensive clinical management ( icm / icm ) , acute ipsrt and maintenance icm ( ipsrt / icm ) , or acute icm and maintenance ipsrt ( icm / ipsrt ) .
patients were seen weekly during the acute phase and then progressed to biweekly and finally monthly sessions during the maintenance phase .
therapy lasted approximately 55 minutes , while icm sessions , which focused primarily on psychoeducation about bipolar disorder and addressing any issues with medication side effects , were roughly 25 minutes in duration .
initial analyses revealed no differences among conditions in terms of time to stabilization , likely due to the strong pharmacological impact on time to remission .
after controlling for significant covariates of survival time ( marital status , significant medical comorbidities , and comorbid anxiety disorders ) we found that individuals who received acute ipsrt survived longer without a new episode , regardless of the nature of their maintenance treatment ( p=0.01 ) .
patients who received acute ipsrt achieved significantly higher regularity of social rhythms than those individuals assigned to acute icm ( p0.001 ) and the degree of protection that ipsrt subjects received from the therapy was correlated with the extent of increase in their social routines ( p0.05 ) . from this study
, we concluded that ipsrt was an effective adjunct to the pharmacological treatment of bipolar disorder , primarily in the preventative capacity .
ipsrt was also studied as one of three intensive psychosocial treatments in the systematic treatment enhancement program for bipolar disorder ( stepbd).this multisite investigation involved 15 different academic centers in the united states and examined four disorder - specific psychosocial approaches to the treatment of bipolar disorder in conjunction with protocol - driven pharmacotherapy on time to recovery and the likelihood of remaining well following an episode of bipolar depression .
a total of 293 individuals with bipolar i or ii disorder were randomly allocated to intensive psychotherapy ( n=163 ) or collaborative care ( cc , n=130 ) , a brief psychoeducational intervention .
intensive psychotherapy was given weekly and then biweekly for up to 30 sessions over 9 months , according to the manuals for family - focused therapy ( fft ) , ipsrt , or cognitive - behavioral therapy ( cbt ) .
cc consisted of three face - to - face sessions over 6 weeks and the provision of a workbook and videotape outlining the essential elements of each of the three intensive treatments .
the primary outcomes of interest were time to recovery from depression and the proportions of patients classified as well during each of 12 study months .
patients assigned to intensive psychotherapy had significantly higher year - end recovery rates ( 64% vs 52% ) and shorter times to recovery than did patients in cc control conditions ( hazard ratio = 1.47 ; 95% ci = 1.08 - 2.00 ) .
patients in intensive psychotherapy were 1.58 times ( 95% ci = 1.17 - 2.13 ) more likely to be clinically well during any study month than those in cc .
post - hoc comparisons of the individual intensive therapies to cc indicated significant benefit of ipsrt with respect to time to remission .
there was also an advantage of intensive psychotherapy in terms of improved relational functioning .
ipsrt is implemented in a series of four phases . regardless of the patient 's clinical state at the beginning of treatment ( either in an acute episode or remission ) the first phase of treatment is always a focused history - taking . during this phase , the clinician seeks to establish the correct diagnosis and then to assess the linkage between acute episodes and interpersonal issues and social routines in the patient 's history , thus developing the foundation for treatment .
in addition to taking a detailed history , the clinician also takes the time to provide the patient and involved family members with education about the nature of bipolar mood disorder , being particularly careful to take into consideration what he or she may already know about the illness . also part of this initial phase of treatment
is an information - gathering process that we refer to as the interpersonal inventory ( ii ) . through this semistructured interview
, the therapist assesses the nature and quality of the patient 's current and past interpersonal relationships .
once these evaluations have been made , the clinician then proceeds to appraising the regularity of the patient 's social routines by using the srm .
the initial phase concludes with the selection of an interpersonal problem area ( unresolved grief , role transition , role disputes , or more pervasive interpersonal deficits ) , upon which the patient and the clinician both agree .
the combination of the history - taking and the ii should allow the clinician and patient to reach a consensus about which of these four problem areas is most closely linked to the onset of the most recent mood episode .
this focus then , becomes the initial jumping - off point for the interpersonal part of the therapy .
depending upon the severity and duration of the patient 's past psychiatric history , as well as the complexity of the patient 's current interpersonal relationships and level of insight into his or her own illness , this initial phase of treatment can last anywhere from three to five sessions .
once the first phase of treatment is completed , the clinician moves on to the second or intermediate phase of therapy .
the focus of this phase of the intervention involves helping the patient establish more regular dally social routines and resolve the interpersonal problem area specified in the initial phase of treatment . during this phase of ipsrt
however , depending upon the patient 's clinical status , more or less frequent sessions may be more appropriate .
in the two large trials of ipsrt conducted to date , this phase has typically required 10 to 12 sessions ; however , in a small open study of bipolar disorder complicated by full - criterion borderline personality disorder , a much longer intermediate phase - of the order of 9 to 10 months - was required to achieve mood stability .
the next phase of treatment , continuation or maintenance ipsrt , focuses on building up patients ' confidence in their capability to use the skills learned in the acute phase of treatment to maintain their current euthymic mood , level of functioning , and social rhythm regularity .
the objective is for the patient to be able to maintain regular social rhythms despite the probable occurrence of stressors such as job changes , vacations , and other unexpected life events .
additionally , the patient is encouraged to continue to improve the quality of his or her interpersonal relationships and keep the level of interpersonal distress at a minimum .
techniques that are commonly used for accomplishing these interpersonal goals include communication analysis , which allows the therapist and patient to identify problem areas in communication to help the patient interact more effectively with significant others ; role - play , which allows the patient a safe environment in which to practice expressing emotions and self - assertion ; and decision analysis , which helps patients to reflect on the potential risks and benefits of alternate choices and options with regard to a specific problem .
more detailed explanations of ipt techniques and strategies can be found in the manual for interpersonal psychotherapy .
treatment frequency generally decreases from weekly , to biweekly , and eventually to monthly sessions as the patient moves from acute to maintenance therapy .
when termination of treatment is deemed appropriate ( or is otherwise necessitated by outside factors such as financial constraints or logistical challenges ) the clinician and patient will begin work on the final phase of treatment which focuses on impending termination .
this can typically be accomplished within three to five monthly sessions . if it is not considered clinically appropriate to stop treatment completely , this final phase can also be utilized to further decrease the frequency of sessions to little more than occasional booster sessions when necessary .
ipsrt can also be successfully implemented simply as a short - term treatment , in which case the initial phase is more condensed and the work on the interpersonal problem areas must be more concentrated . despite the shortened overall duration of treatment in this case , it is still advisable that treatment frequency be reduced near the end , so as to still allow for at least three to four bimonthly sessions to accomplish the necessary termination steps .
the following is a detailed example of how ipsrt is utilized in the treatment of a patient with bipolar i disorder .
anne is a 35-year - old separated woman who began ipsrt while in the throes of a particularly severe depression that had had its onset 4 months previously .
anne had been working as a waitress at a local restaurant , with a schedule that varied widely : she never had a consistent day off , and she was frequently scheduled for lunchtime shifts one day followed by evening shifts the next . she had recently moved out the home she shared with her estranged husband and she was having great difficulty making ends meet , with the rental fees of her new apartment and the need to buy a car of her own .
as anne 's depression worsened , she found herself struggling with disrupted sleep and a lack of motivation which began to negatively impact her work .
she began showing up late for her shifts , and the fatigue stemming from her erratic sleep schedule caused her to make several mistakes with her customers .
soon she was in serious jeopardy of losing her job , the prospect of which sparked intense financial anxiety .
while anne had a rather protracted history of brief but severe manias , this appeared to be one of very few depressions anne had experienced in her life .
additionally , anne had married young ( at age 20 ) and had , for all intents and purposes , moved directly from her parents ' home into the home she shared with her husband .
she had never actually lived alone , and was finding her new circumstances as frightening as they were difficult .
anne 's therapist could see that there were several factors that would need to be addressed in order to help anne out of her depression .
first , anne 's schedule was far too erratic . in order for anne to be able to regulate her daily routines , there had to be a routine established to begin with .
second , it appeared to the clinician that anne 's primary interpersonal problem was the role transition from being a married woman to a single one and the challenges of self - sufficiency that came along with it .
the particularly stressful life event of her marital separation was also the impetus for her current disordered state of affairs , so work focused on ameliorating her interpersonal stress would be fundamental to achieving stable social rhythms . once anne 's therapist had completed the history - taking and interpersonal inventory , she and anne moved into the intermediate phase of treatment .
anne 's therapist first began problem - solving with anne about how to make her schedule more consistent at work .
after discussing the nature of anne 's relationship with her supervisor ( good until she had started missing work ) , her therapist suggested she talk with her supervisor about requesting set shifts on a weekly basis .
while anne knew that , because of the nature of the restaurant business , it would be nearly impossible for her to have the same days off each week , she agreed that her boss might be somewhat receptive to the idea of at least making her shifts take place during the same times each day , especially if it meant this would help her to be a more reliable employee . with anne 's history of mania ,
her therapist suggested that she avoid the late - night shifts if at all possible .
anne 's supervisor was in fact amenable to her requests , and upon successfully obtaining a more stable work schedule , anne and her therapist then went to work on regulating her sleep schedule .
using the srm as a guide , anne and her therapist agreed on set times when anne would go to bed at night and get up in the morning , aiming to have these times vary by no more than an hour , even on her days off .
anne 's therapist offered her education on sleep hygiene , and explained how getting better sleep would not only help her mood , but would also make her less clumsy and forgetful at work , thereby alleviating some of her work - related stress and worry . while this behavioral work was being done to help regulate anne 's social rhythms , her therapist was simultaneously working with her on her role transition to being a single woman and dealing with the stress and loneliness she felt as a result of her marital separation . her therapist stressed the importance of creating a solid support network to help her through this difficult time , encouraging anne to find ways to express her feelings about her current situation .
anne 's therapist knew from the ii that anne maintained a good relationship with her parents and had at least two female friends from high school with whom she remained close ; however , she rarely saw either her parents or these girlfriends because she felt too depressed to leave her apartment other than to drag herself to work .
she encouraged anne to visit her parents on one of her days off and to make some arrangements to see one of her friends on the other day .
anne noted a link between getting herself to her parents ' house and improvement in her mood .
she also found that after doing something with her friends , she felt better as well .
as anne continued to work on regularizing her daily routines and improving her satisfaction with her interpersonal relationships , she felt her depressive symptoms begin to dissipate .
anne 's therapist remained cognizant of anne 's history of mania , and kept a careful eye out for any signs that anne 's mood disorder was not actually remitting , but rather was cycling into an episode of mania .
she stressed that it was important for anne to not become too overstimulated ( especially considering the often hectic nature of her job ) , in the hopes of preventing a manic recurrence .
anne remained in the acute phase of treatment for approximately 22 weeks , and then moved into the maintenance phase of treatment .
after three biweekly sessions , anne and her therapist moved to monthly sessions where they focused on maintaining anne 's routine regularity and strove to stay ahead of any potential pitfalls to her progress , particularly the stress of impending divorce proceedings .
anne is a 35-year - old separated woman who began ipsrt while in the throes of a particularly severe depression that had had its onset 4 months previously .
anne had been working as a waitress at a local restaurant , with a schedule that varied widely : she never had a consistent day off , and she was frequently scheduled for lunchtime shifts one day followed by evening shifts the next .
she had recently moved out the home she shared with her estranged husband and she was having great difficulty making ends meet , with the rental fees of her new apartment and the need to buy a car of her own .
as anne 's depression worsened , she found herself struggling with disrupted sleep and a lack of motivation which began to negatively impact her work .
she began showing up late for her shifts , and the fatigue stemming from her erratic sleep schedule caused her to make several mistakes with her customers .
soon she was in serious jeopardy of losing her job , the prospect of which sparked intense financial anxiety .
the therapist 's detailed history - taking revealed the following information . while anne had a rather protracted history of brief but severe manias , this appeared to be one of very few depressions anne had experienced in her life .
additionally , anne had married young ( at age 20 ) and had , for all intents and purposes , moved directly from her parents ' home into the home she shared with her husband .
she had never actually lived alone , and was finding her new circumstances as frightening as they were difficult .
anne 's therapist could see that there were several factors that would need to be addressed in order to help anne out of her depression .
first , anne 's schedule was far too erratic . in order for anne to be able to regulate her daily routines
second , it appeared to the clinician that anne 's primary interpersonal problem was the role transition from being a married woman to a single one and the challenges of self - sufficiency that came along with it .
the particularly stressful life event of her marital separation was also the impetus for her current disordered state of affairs , so work focused on ameliorating her interpersonal stress would be fundamental to achieving stable social rhythms . once anne 's therapist had completed the history - taking and interpersonal inventory , she and anne moved into the intermediate phase of treatment .
anne 's therapist first began problem - solving with anne about how to make her schedule more consistent at work .
after discussing the nature of anne 's relationship with her supervisor ( good until she had started missing work ) , her therapist suggested she talk with her supervisor about requesting set shifts on a weekly basis .
while anne knew that , because of the nature of the restaurant business , it would be nearly impossible for her to have the same days off each week , she agreed that her boss might be somewhat receptive to the idea of at least making her shifts take place during the same times each day , especially if it meant this would help her to be a more reliable employee . with anne 's history of mania ,
her therapist suggested that she avoid the late - night shifts if at all possible .
anne 's supervisor was in fact amenable to her requests , and upon successfully obtaining a more stable work schedule , anne and her therapist then went to work on regulating her sleep schedule .
using the srm as a guide , anne and her therapist agreed on set times when anne would go to bed at night and get up in the morning , aiming to have these times vary by no more than an hour , even on her days off .
anne 's therapist offered her education on sleep hygiene , and explained how getting better sleep would not only help her mood , but would also make her less clumsy and forgetful at work , thereby alleviating some of her work - related stress and worry . while this behavioral work was being done to help regulate anne 's social rhythms , her therapist was simultaneously working with her on her role transition to being a single woman and dealing with the stress and loneliness she felt as a result of her marital separation .
her therapist stressed the importance of creating a solid support network to help her through this difficult time , encouraging anne to find ways to express her feelings about her current situation .
anne 's therapist knew from the ii that anne maintained a good relationship with her parents and had at least two female friends from high school with whom she remained close ; however , she rarely saw either her parents or these girlfriends because she felt too depressed to leave her apartment other than to drag herself to work .
she encouraged anne to visit her parents on one of her days off and to make some arrangements to see one of her friends on the other day .
anne noted a link between getting herself to her parents ' house and improvement in her mood .
she also found that after doing something with her friends , she felt better as well .
as anne continued to work on regularizing her daily routines and improving her satisfaction with her interpersonal relationships , she felt her depressive symptoms begin to dissipate .
anne 's therapist remained cognizant of anne 's history of mania , and kept a careful eye out for any signs that anne 's mood disorder was not actually remitting , but rather was cycling into an episode of mania .
she stressed that it was important for anne to not become too overstimulated ( especially considering the often hectic nature of her job ) , in the hopes of preventing a manic recurrence .
anne remained in the acute phase of treatment for approximately 22 weeks , and then moved into the maintenance phase of treatment .
after three biweekly sessions , anne and her therapist moved to monthly sessions where they focused on maintaining anne 's routine regularity and strove to stay ahead of any potential pitfalls to her progress , particularly the stress of impending divorce proceedings .
ipsrt has been supported empirically through two large studies involving the therapy in combination with pharmacotherapeutic interventions in the treatment of bipolar disorder .
the first of these studies involved 175 patients with bipolar i disorder who presented for treatment while in the midst of a depressive , manic , or mixed episode .
in this two - phase study , these individuals were randomly allocated to four acute and maintenance treatment sequences : acute and maintenance ipsrt ( ipsrt / ipsrt ) , acute and maintenance intensive clinical management ( icm / icm ) , acute ipsrt and maintenance icm ( ipsrt / icm ) , or acute icm and maintenance ipsrt ( icm / ipsrt ) .
patients were seen weekly during the acute phase and then progressed to biweekly and finally monthly sessions during the maintenance phase .
therapy lasted approximately 55 minutes , while icm sessions , which focused primarily on psychoeducation about bipolar disorder and addressing any issues with medication side effects , were roughly 25 minutes in duration .
initial analyses revealed no differences among conditions in terms of time to stabilization , likely due to the strong pharmacological impact on time to remission .
after controlling for significant covariates of survival time ( marital status , significant medical comorbidities , and comorbid anxiety disorders ) we found that individuals who received acute ipsrt survived longer without a new episode , regardless of the nature of their maintenance treatment ( p=0.01 ) .
patients who received acute ipsrt achieved significantly higher regularity of social rhythms than those individuals assigned to acute icm ( p0.001 ) and the degree of protection that ipsrt subjects received from the therapy was correlated with the extent of increase in their social routines ( p0.05 ) . from this study
, we concluded that ipsrt was an effective adjunct to the pharmacological treatment of bipolar disorder , primarily in the preventative capacity .
ipsrt was also studied as one of three intensive psychosocial treatments in the systematic treatment enhancement program for bipolar disorder ( stepbd).this multisite investigation involved 15 different academic centers in the united states and examined four disorder - specific psychosocial approaches to the treatment of bipolar disorder in conjunction with protocol - driven pharmacotherapy on time to recovery and the likelihood of remaining well following an episode of bipolar depression .
a total of 293 individuals with bipolar i or ii disorder were randomly allocated to intensive psychotherapy ( n=163 ) or collaborative care ( cc , n=130 ) , a brief psychoeducational intervention .
intensive psychotherapy was given weekly and then biweekly for up to 30 sessions over 9 months , according to the manuals for family - focused therapy ( fft ) , ipsrt , or cognitive - behavioral therapy ( cbt ) .
cc consisted of three face - to - face sessions over 6 weeks and the provision of a workbook and videotape outlining the essential elements of each of the three intensive treatments .
the primary outcomes of interest were time to recovery from depression and the proportions of patients classified as well during each of 12 study months .
patients assigned to intensive psychotherapy had significantly higher year - end recovery rates ( 64% vs 52% ) and shorter times to recovery than did patients in cc control conditions ( hazard ratio = 1.47 ; 95% ci = 1.08 - 2.00 ) .
patients in intensive psychotherapy were 1.58 times ( 95% ci = 1.17 - 2.13 ) more likely to be clinically well during any study month than those in cc .
post - hoc comparisons of the individual intensive therapies to cc indicated significant benefit of ipsrt with respect to time to remission .
there was also an advantage of intensive psychotherapy in terms of improved relational functioning .
a small , but consistent , body of data now suggests that an intervention designed to regularize patients ' social rhythms , and presumably thereby their circadian rhythms , has significant positive effects on the course of bipolar disorder . in our original maintenance trial ,
that effect was observed for the impact of acute treatment on long - term survival without a new mood episode , while in the step - bd trial acute ipsrt had a positive effect on time to remission of bipolar depression .
additional studies are under way to explore ipsrt as treatments for bipolar disorder in adolescents and as monotherapy for bipolar ii disorder . | bipolar disorder is characterized by frequent recurrences , often related to noncompliance with drug treatment , stressful life events , and disruptions in social rhythms .
interpersonal and social rhythm therapy ( ipsrt ) was designed to directly address these problem areas .
this article discusses the circadian basis of ipsrt and the importance of stable daily routines in the maintenance of the euthymic state , as well as the two large controlled trials which empirically support this intervention .
the authors discuss the advantages of ipsrt as an acute intervention , as well as a prophylactic treatment for both bipolar i and ii disorder . using a case example ,
the authors describe how ipsrt is implemented in a clinical setting , detailing the therapeutic methods and processes involved . |
alcohol plays a significant role in accidents , injuries , and their outcomes . according to the world health organization ( who ) , there are 76.3 million people with alcohol use disorders worldwide ;
in 2000 , 1.8 million deaths and loss of 58.3 million disability - adjusted life years were attributed to alcohol .
although the association between alcohol consumption and trauma - related morbidity and mortality is well - documented , particularly in the us , there is much less data on trauma and alcohol in poland and other eastern european countries . in this paper
we systematically review and summarize findings of the research published in the last 10 years on injury and alcohol in poland .
then , we summarize research on alcohol - related injuries , focusing on intentional and unintentional injuries . finally , we suggest recommendations for future training in emergency medical settings to address this serious and growing problem .
a comprehensive search of polish- and english - language journal articles was conducted using electronic databases including pubmed and medline , and web - based sources such as emedicine .
additional information such as statistics related to injury and alcohol consumption was derived from polish governmental and police publications , the world health organization ( who ) , and organization for economic co - operation and development ( oecd ) reports .
the search was conducted using the following keywords : alcohol / ethanol , poland , trauma , injury , violence , traffic , suicide , gender , and alcohol consumption . from pubmed and medline searches ,
thirty - one articles were found to fit the criteria , and 42 articles were excluded because they focused on alcohol as a risk factor for other health problems such as stroke , cancer , or cardiovascular disease .
moskalewicz presented five indicators of the increase in alcohol consumption immediately following the privatization of the alcohol industry in the late 1980s : increases in the rates of hospital admissions for alcoholic psychoses , hospital admissions due to alcohol dependence , psychiatric hospital admissions due to alcohol - related disorders ( dependence and psychosis ) , increase in the rate of liver cirrhosis mortality , and increasing alcohol - related road accidents .
alcohol sales data show that consumption leveled off in the late 1990s , but has been increasing since the beginning of this century .
although distilled spirits and liquor are the preferred alcoholic beverages in poland , beer consumption has steadily increased [ 4 , 5 ] . in 2003 , beer consumed in pure alcohol units was 3.95 l per capita , and spirits consumed in pure alcohol units was 1.3 l per capita .
the region of central and eastern europe has among the highest levels of alcohol consumption in the world .
russia was found to have the most detrimental drinking patterns compared to eu member states such as hungary , lithuania , poland , and the czech republic , while the czech republic had the highest overall beer consumption levels in europe , but a relatively less detrimental pattern of drinking . the estimated average pattern of drinking of 14 drinks , with 4 being the most detrimental pattern and mainly based on heavy drinking and drinking to intoxication per occasion . france and sweden were found to differ both in volume of alcohol consumption and in drinking patterns , and the uk was found to have a beer - drinking culture , with a rise in heavy irregular drinking in recent years .
recently published research based on surveys and data from the world health organization ( who ) establishes yearly premature alcohol - attributable traumatic deaths in adolescents in poland at over 6,000 , with 4,610 unintentional and 1,478 intentional injuries .
all alcohol - attributable deaths in poland are estimated at about 11,000 cases a year with a death rate of 8.7 per 10,000 for males and 0.9 per 10,000 for females . although comparable to other eastern european countries , it is higher than in other western european countries , such as sweden , which has 2.7 and 0.5 deaths per 10,000 for men and women , respectively , and in the us , which is estimated to have a total overall death rate per 10,000 of 2.7 . in 2007 ,
the standarized death rate ( sdr , per 100,000 people ) in poland was 13.45 for motor vehicle traffic accidents , 15.73 for suicide and intentional self - harm injuries , and 1.31 for homicide and assaults ; however , statistics on how many of these deaths are attributed to alcohol are not available .
hospital - based data illustrate a high burden of heavy drinking and alcohol dependence in poland . in a primary care study , nearly 64% ( n = 4,373 ) of patients seeking routine health care had consumed alcohol in the previous 3 months .
of these , 19% were dependent drinkers ( three or more positive cage responses and/or those who consumed five or more drinks per day ) , about 36% of men reported more than 21 drinks per week , and about 21% of women had more than 14 drinks per week .
studies show differences in alcohol consumption by gender , age , education , and region within poland .
9% of women consumed an average of more than 34 drinks / day ( > 40 g / day ) , while 16.4% of men and 34.3% of women consumed very little or were abstinent . in another study ,
almost 50% of alcohol - dependent patients ( n = 458 ) aged 1864 reported drinking more than 4 times / week .
beer was the most frequently used beverage ( 67.4% ) , followed by vodka ( 63.2% ) and wine ( 39.5% ) .
studies report that higher education among women is related to increased alcohol consumption [ 13 , 15 , 18 ] , and women living in central / urban settings may consume as much as 50% more than women in rural settings .
similar to other countries , including the us , a large percentage of the youth engage in hazardous drinking patterns , such as binge drinking [ 1922 ] . among 1516-year - old students in 35 european countries ,
heavy episodic drinking during the past 30 days increased between 19951999 and between 20032007 , especially among girls .
between 20032007 , the proportion of students reporting heavy episodic drinking during the past 30 days increased by 16% in poland , while the highest increase of 31% occurred in portugal .
men in the us are more likely to report binge drinking than women , and in 2006 , binge and heavy drinking were highest for the 18- to 25-year - old group compared to other age groups , with the peak rate occurring at 2123 years .
comparison of drinking rates among 1516 years olds between european countries and the us shows that the us is a low consumption country by european standards .
poland had a higher prevalence rate ( 65% ) of drinking in the past 30 days than the us ( 35% ) , with the highest rate being in austria ( 82% ) .
the percentage of young people in the us who reported binge drinking was lower ( 22% ) than all other european countries except turkey ( 15% ) .
screening of two polish samples ( 18 years and older ) seeking emergency services revealed males were more likely to be younger , be injured , and meet criteria for alcohol use disorders than females . among those who drank , males were more likely to report weekly drinking and 5 + drinks on an occasion than women , and nearly 50% of young women reported binge drinking .
there is also a higher incidence of reported alcohol - related mortality in men compared to women [ 4 , 13 ] , and more alcohol - related problems ( such as difficulties with marriage / partner , friends / social life , work , physical and mental health , finance , police and other authorities ) .
although few studies in poland have addressed psychosocial factors associated with alcohol misuse , one study found that a perceived imbalance in the effort - to - reward ratio at work was associated with binge drinking , increased overall alcohol intake , and problem drinking .
patterns of alcohol use among adolescents have changed as well . a study of alcohol consumption among adolescents found that rates of heavy drinking increased between 1988 and 2004 . in this study , both the type and quantity of alcohol consumed increased , reflecting shifts in the political , economic and cultural environment . with the rapid development of hospital emergency departments ( eds ) and emergency medicine as a specialty in poland ,
rates for all injuries ( intentional and unintentional ) in poland are higher than in the rest of the european union ( eu ) .
alcohol is one of the top ten health and injury risk factors for men and women .
a number of studies conducted among injured ed / trauma patients in poland have shown a positive association between alcohol consumption and injury [ 4 , 14 , 16 , 2931 ] .
a case - crossover analysis of emergency services patients from warsaw and sosnowiec showed a four - fold risk of any injury for those drinking within 6 h compared to those who had not been drinking in both the samples .
men are more likely than women to be injured from drinking directly prior to the injury .
males who drank more than 12 l of alcohol annually were 2.4 times more likely to sustain injuries than those who drank less than 12 l annually .
injured men were twice as likely to report drinking within 6 h before the injury event than non - injured men presenting to the ed .
michalska et al . found alcohol intoxication to be a debilitating factor in prompt diagnosis , assessment , and treatment of cranio - cerebral injury .
in addition to examining overall injury rates , it is important to determine patterns of unintentional and intentional injury to be able to develop strategies to best deal with the complex interaction of alcohol and injury in the region .
unintentional injury is the fourth leading cause ( 12.8% ) of disability in males and seventh leading cause in females ( 4.8% ) in poland .
who / europe and the european commission noted that the leading causes of unintentional injury death are transportation injuries , followed by falls , poisoning , drowning , and fires .
there are very few studies conducted in poland that focus on unintentional injuries other than traffic injuries .
one study examined 1,199 patients ( 1479 years ) visiting the department of surgery at a university hospital in krakow from 19872000 for treatment of hand injuries .
of those , 320 patients ( 26.7% ) were found to be under the influence of alcohol at the time of injury , and most of them ( 89.3% ) were males and manual workers ( 74.3% ) . around 48.4% of patients suffered from injuries caused by
a cut with glass , and 5.6% had operated various kinds of machines and mechanical equipment under the influence of alcohol .
most accidents happened at home ( 65.9% ) , while the others occurred in public places ( 15.9% ) , in the street ( 11.8% ) , on a farm ( 3.1% ) , and at the workplace ( 1.25% ) .
autopsies done in a study investigating the cause of mortality among polish small - scale fishermen during a 40-year period reports finding > 0.5% ( 0.5
g / l ) blood alcohol levels in 45% of deaths out of the 82 cases where autopsy was carried out ( n = 177 ) .
alcohol use was identified in 7 sea catastrophe - related cases and in 30 cases connected with other reasons for drowning with the greatest risk related to mooring , hauling , and boarding the nets .
the authors report that alcohol may be implicated in at least one - quarter of the accidents and may have contributed to other factors , such as loss of balance on deck and injury preceding falling or being swept overboard . in 2003 , 11% of all traffic accidents in poland were attributed to alcohol ; alcohol was associated with death in 13% of all traffic - related mortalities .
police reports revealed 167,318 drunk - driving cases in 2003 , out of which 18,830 of the drivers had a bac of 0.20.5 ml / g , while 148,488 had a bac of more than 0.5
the study at the university hospital in krakow from 19872000 reported that 7.1% ( n = 320 ) of those found to be under the influence of alcohol at the time of treatment for hand injuries were injured in traffic accidents .
the rates of homicide in poland are higher than in the rest of the eu .
intentional injury is also the sixth leading cause ( 4.8% ) of disability in males .
studies conducted specifically in poland , as well as those conducted across several other countries , have shown that alcohol consumption often precedes violent events [ 30 , 3841 ] . in samples of ed patients from warsaw and sosnowiec , poland , cherpitel et al .
( 2005 ) found that there was over a 17-fold increased risk for a violence - related injury for individuals who reported drinking 6 h before the injury , the risk being greater for women than for men .
an epidemiological investigation of the prevalence of potentially traumatic events ( ptes ) in a sample of alcohol - dependent patients in poland found that the numbers of ptes reported were related to higher post - traumatic stress , but not necessarily to higher alcohol use intensity . a higher level of alcohol misuse was associated with two categories of ptes : robbery and mugging , and witnessing death or assault .
alcohol has been found to be a strong risk factor for intimate partner violence in poland .
analyzed data from a survey of 800 participants ( 400 perpetrators and their 400 victims of domestic violence ) in the office of the association for violence prevention in the city of lublin , poland .
results suggested that perpetrators with a history of alcohol abuse were more likely to be younger , had less education , were less likely to be permanently employed , and had more problems with the legal system than perpetrators without a history of alcohol abuse . in the same sample , 76% of domestic violence perpetrators had problems related to alcohol , as measured by an audit score > 8 .
in addition , another study in the country found 25% of surveyed pregnant women reported physical and emotional abuse by their partners .
men that physically abused pregnant women in this study were more often found to have primary school education , be unemployed , and drink alcohol .
a substantial number of 15 to 20 year olds in poland reported having experienced at least one of four kinds of parent - child violence ( emotional , physical , negligence , and sexual abuse ) , of which parental alcohol abuse was one of the most frequently cited reasons for family violence . in poland , suicide is the leading cause of intentional injury - related death , and suicide rates are higher than in the rest of the eu . from 1989 to 1994 , a time of rapid social and political change , the suicide rate in poland increased by 26.5% .
the study aimed at describing suicide mortality in eastern europe by investigating the covariation between changes in some of the hypothesized causes such as alcohol consumption , economic situation , general social stress , political situation , societal ( dis)organization , and the changes in the suicide rates . the linear regression model , which consisted of variables such as hypothetical general stress ( as indicated by mortality / life expectancy ) , democratization , alcohol consumption , and social disorganization ( with a period - dependent effect ) , was able to predict percentual changes in the suicide rates in 16 out of the 28 eastern bloc countries from 19841989 and 19891994 fairly accurately , but failed to do so for poland besides other countries .
on the other hand , landberg ( 2008 ) noted that the relationship between alcohol consumption and suicide mortality tends to be strongest in countries with detrimental drinking patterns , such as poland ( based on who s hazardous patterns score ) . in this cross - cultural study that included poland , a 1-l increase in per capita consumption of alcohol
was associated with an increase in overall suicide rates by 5.77.5% , with males at higher risk than females . in another study , among 154 alcohol - dependent patients admitted to addiction treatment centers in poland , 43% reported attempting suicide at some point during their lifetime .
post - mortem reports have shown the presence of alcohol in almost 50% of polish suicide victims . given the strong association between alcohol consumption and injury in poland , it is imperative that medical professionals be prepared to respond .
academic emergency departments , the polish emergency medicine society , and psychology and social service societies , among other entities , will be key in the development and delivery of training materials to emergency , primary care , social service , and addiction treatment professionals . based on training recommendations for the us and other countries , training of current and future ed physicians , nurses , emts , social workers , and psychologists in poland and eastern europe in general should include the following areas :
recognition and assessmentto ensure that clinicians are knowledgeable in assessing the quantity and frequency of alcohol and other drug use among patients seen in medical and social service settings , key recommendations include : ( 1 ) increasing preclinical and clinical training regarding ( a ) the available alcohol screening instruments ; ( b ) methods to determine other medication and drug use [ including over - the - counter ( otc ) medications and herbal agents ] ; ( c ) techniques for conducting screening ; ( d ) techniques to incorporate structured screening for alcohol , medications , and illicit drugs into standard emergency practice ; and ( 2 ) developing preclinical and clinical training evaluations and tests that include questions on substance use screening.a study comparing performance of alcohol screening instruments to identify alcohol use disorders in the emergency services setting in the warsaw and sosnowiec regions of poland found sensitivity of the raps4 and alcohol use disorders identification test ( audit ) to be significantly better than the cage for alcohol dependence among males in warsaw , but specificity to be poorer in males in both the samples .
the cage appeared to perform considerably better for both males and females in sosnowiec than in warsaw . among females ,
sensitivity for alcohol abuse / harmful drinking and for alcohol dependence or abuse / harmful drinking was significantly better for the raps4-qf than for the cage or audit at a cutoff point of 8 across both sites .
however , a smaller - scale research study found that the audit questionnaire was more sensitive than the cage when implemented in polish emergency departments .
this finding is supported by a number of studies in the us and uk in a variety of medical settings [ 5053 ] .
the audit has been found to have good psychometric properties in a number of countries with a variety of patient populations.conducting structured and targeted brief advice / interventionsadvice and brief interventions can be delivered as part of early detection programs in eds and other medical and psychosocial treatment settings .
brief alcohol interventions such as tailored or generic booklets with brief counseling or motivational interviewing with an interventionist have been proven to be effective in a variety of medical settings , including the emergency department [ 5459 ] .
brief interventions have two purposes : ( 1 ) to help individuals who are classified as at - risk for alcohol problems to cut back or stop using alcohol or drugs , and ( 2 ) to help individuals with more serious substance use problems engage in needed treatment .
educational materials are available through the national institute on alcohol abuse and alcoholism ( niaaa ) and through the center for substance abuse treatment ( csat ) in the treatment improvement protocol ( tip ) # 34 ,
brief interventions and brief therapies for substance abuse , as well as through the sbirt ( screening , brief intervention , referral , and treatment ) quick guide .training can be made available to professionals who work in emergency care , primary care , and psychosocial programs through preclinical and clinical education programs , continuing education credits , and other workshop opportunities .
these are needed because , in a changing health care delivery system , brief , effective methods to deal with alcohol problems will be both clinically and cost effective .
one such short , concentrated training session at yale university ed in connecticut significantly improved knowledge and practice of screening and interventions for alcohol problems among a sample of emergency medicine residents , while the control group at harvard university in massachusetts showed no changes .
there are very few preclinical and clinical programs that currently include any of the techniques of brief alcohol interventions in poland.because the ed is a very fast - paced , clinically demanding venue , training personnel in how to identify and manage patients with at - risk and problem drinking should also be accompanied by research on methods to best implement some of the newer , innovative technologies for screening and intervening .
the use of computerized tailored interventions represents one such technique to provide targeted , individualized feedback to injured and medical ed patients .
other technologies that may be useful in the future in poland include the use of interactive voice recognition ( ivr ) technology to facilitate screening , delivery of educational interventions , and follow - up on patient progress over the telephone .
interactive computer programs on laptops or palmtops ; web - based interventions ; computerized bundling of brief health messages for multiple health risks ( e.g. , smoking , alcohol use , seat belt use ) ; and audio interventions tailored to specific problems and delivered through headsets are all potential approaches in emergency , urgent care , and primary care settings .referral to treatmentalso , providers need to have knowledge of the available in- and outpatient programs to help those individuals who meet criteria for more serious substance use problems .
the brief intervention structure provides the opportunity to work with patients who have more serious alcohol problems and to help them consider obtaining formal substance abuse treatment.it is ideal to have psychiatric emergency services co - located in the ed with acute medical and injury care whenever possible to address patients who screen positive for risky alcohol use . alternatively increased collaboration and consultation between ed and mental health services is critical to any successful screening and brief intervention program
. however , specialty resources are limited and often can not be provided during the acute care time frame .
the training recommendations provided are focused on the many eds and clinicians that do not have access or availability to provide this service by specialty psychiatric services .
recognition and assessment to ensure that clinicians are knowledgeable in assessing the quantity and frequency of alcohol and other drug use among patients seen in medical and social service settings , key recommendations include : ( 1 ) increasing preclinical and clinical training regarding ( a ) the available alcohol screening instruments ; ( b ) methods to determine other medication and drug use [ including over - the - counter ( otc ) medications and herbal agents ] ; ( c ) techniques for conducting screening ; ( d ) techniques to incorporate structured screening for alcohol , medications , and illicit drugs into standard emergency practice ; and ( 2 ) developing preclinical and clinical training evaluations and tests that include questions on substance use screening .
a study comparing performance of alcohol screening instruments to identify alcohol use disorders in the emergency services setting in the warsaw and sosnowiec regions of poland found sensitivity of the raps4 and alcohol use disorders identification test ( audit ) to be significantly better than the cage for alcohol dependence among males in warsaw , but specificity to be poorer in males in both the samples .
the cage appeared to perform considerably better for both males and females in sosnowiec than in warsaw . among females ,
sensitivity for alcohol abuse / harmful drinking and for alcohol dependence or abuse / harmful drinking was significantly better for the raps4-qf than for the cage or audit at a cutoff point of 8 across both sites .
however , a smaller - scale research study found that the audit questionnaire was more sensitive than the cage when implemented in polish emergency departments .
this finding is supported by a number of studies in the us and uk in a variety of medical settings [ 5053 ] .
the audit has been found to have good psychometric properties in a number of countries with a variety of patient populations . conducting structured and targeted brief advice / interventions advice and brief interventions
can be delivered as part of early detection programs in eds and other medical and psychosocial treatment settings .
brief alcohol interventions such as tailored or generic booklets with brief counseling or motivational interviewing with an interventionist have been proven to be effective in a variety of medical settings , including the emergency department [ 5459 ] .
brief interventions have two purposes : ( 1 ) to help individuals who are classified as at - risk for alcohol problems to cut back or stop using alcohol or drugs , and ( 2 ) to help individuals with more serious substance use problems engage in needed treatment .
educational materials are available through the national institute on alcohol abuse and alcoholism ( niaaa ) and through the center for substance abuse treatment ( csat ) in the treatment improvement protocol ( tip ) # 34 , brief interventions and brief therapies for substance abuse , as well as through the sbirt ( screening , brief intervention , referral , and treatment ) quick guide .
training can be made available to professionals who work in emergency care , primary care , and psychosocial programs through preclinical and clinical education programs , continuing education credits , and other workshop opportunities .
these are needed because , in a changing health care delivery system , brief , effective methods to deal with alcohol problems will be both clinically and cost effective .
one such short , concentrated training session at yale university ed in connecticut significantly improved knowledge and practice of screening and interventions for alcohol problems among a sample of emergency medicine residents , while the control group at harvard university in massachusetts showed no changes .
there are very few preclinical and clinical programs that currently include any of the techniques of brief alcohol interventions in poland . because the ed is a
very fast - paced , clinically demanding venue , training personnel in how to identify and manage patients with at - risk and problem drinking should also be accompanied by research on methods to best implement some of the newer , innovative technologies for screening and intervening .
the use of computerized tailored interventions represents one such technique to provide targeted , individualized feedback to injured and medical ed patients .
other technologies that may be useful in the future in poland include the use of interactive voice recognition ( ivr ) technology to facilitate screening , delivery of educational interventions , and follow - up on patient progress over the telephone .
interactive computer programs on laptops or palmtops ; web - based interventions ; computerized bundling of brief health messages for multiple health risks ( e.g. , smoking , alcohol use , seat belt use ) ; and audio interventions tailored to specific problems and delivered through headsets are all potential approaches in emergency , urgent care , and primary care settings .
referral to treatment also , providers need to have knowledge of the available in- and outpatient programs to help those individuals who meet criteria for more serious substance use problems . the brief intervention structure provides the opportunity to work with patients who have more serious alcohol problems and to help them consider obtaining formal substance abuse treatment .
it is ideal to have psychiatric emergency services co - located in the ed with acute medical and injury care whenever possible to address patients who screen positive for risky alcohol use . alternatively increased collaboration and consultation between ed and mental health services is critical to any successful screening and brief intervention program
. however , specialty resources are limited and often can not be provided during the acute care time frame .
the training recommendations provided are focused on the many eds and clinicians that do not have access or availability to provide this service by specialty psychiatric services .
moskalewicz presented five indicators of the increase in alcohol consumption immediately following the privatization of the alcohol industry in the late 1980s : increases in the rates of hospital admissions for alcoholic psychoses , hospital admissions due to alcohol dependence , psychiatric hospital admissions due to alcohol - related disorders ( dependence and psychosis ) , increase in the rate of liver cirrhosis mortality , and increasing alcohol - related road accidents .
alcohol sales data show that consumption leveled off in the late 1990s , but has been increasing since the beginning of this century .
although distilled spirits and liquor are the preferred alcoholic beverages in poland , beer consumption has steadily increased [ 4 , 5 ] . in 2003 , beer consumed in pure alcohol units was 3.95 l per capita , and spirits consumed in pure alcohol units was 1.3 l per capita .
the region of central and eastern europe has among the highest levels of alcohol consumption in the world .
russia was found to have the most detrimental drinking patterns compared to eu member states such as hungary , lithuania , poland , and the czech republic , while the czech republic had the highest overall beer consumption levels in europe , but a relatively less detrimental pattern of drinking . the estimated average pattern of drinking of 14 drinks , with 4 being the most detrimental pattern and mainly based on heavy drinking and drinking to intoxication per occasion . france and sweden were found to differ both in volume of alcohol consumption and in drinking patterns , and the uk was found to have a beer - drinking culture , with a rise in heavy irregular drinking in recent years .
recently published research based on surveys and data from the world health organization ( who ) establishes yearly premature alcohol - attributable traumatic deaths in adolescents in poland at over 6,000 , with 4,610 unintentional and 1,478 intentional injuries .
all alcohol - attributable deaths in poland are estimated at about 11,000 cases a year with a death rate of 8.7 per 10,000 for males and 0.9 per 10,000 for females . although comparable to other eastern european countries , it is higher than in other western european countries , such as sweden , which has 2.7 and 0.5 deaths per 10,000 for men and women , respectively , and in the us , which is estimated to have a total overall death rate per 10,000 of 2.7 . in 2007 ,
the standarized death rate ( sdr , per 100,000 people ) in poland was 13.45 for motor vehicle traffic accidents , 15.73 for suicide and intentional self - harm injuries , and 1.31 for homicide and assaults ; however , statistics on how many of these deaths are attributed to alcohol are not available .
hospital - based data illustrate a high burden of heavy drinking and alcohol dependence in poland . in a primary care study , nearly 64% ( n = 4,373 ) of patients seeking routine health care had consumed alcohol in the previous 3 months .
of these , 19% were dependent drinkers ( three or more positive cage responses and/or those who consumed five or more drinks per day ) , about 36% of men reported more than 21 drinks per week , and about 21% of women had more than 14 drinks per week .
studies show differences in alcohol consumption by gender , age , education , and region within poland .
9% of women consumed an average of more than 34 drinks / day ( > 40 g / day ) , while 16.4% of men and 34.3% of women consumed very little or were abstinent . in another study ,
almost 50% of alcohol - dependent patients ( n = 458 ) aged 1864 reported drinking more than 4 times / week .
beer was the most frequently used beverage ( 67.4% ) , followed by vodka ( 63.2% ) and wine ( 39.5% ) .
studies report that higher education among women is related to increased alcohol consumption [ 13 , 15 , 18 ] , and women living in central / urban settings may consume as much as 50% more than women in rural settings .
similar to other countries , including the us , a large percentage of the youth engage in hazardous drinking patterns , such as binge drinking [ 1922 ] . among 1516-year - old students in 35 european countries ,
heavy episodic drinking during the past 30 days increased between 19951999 and between 20032007 , especially among girls .
between 20032007 , the proportion of students reporting heavy episodic drinking during the past 30 days increased by 16% in poland , while the highest increase of 31% occurred in portugal .
men in the us are more likely to report binge drinking than women , and in 2006 , binge and heavy drinking were highest for the 18- to 25-year - old group compared to other age groups , with the peak rate occurring at 2123 years .
comparison of drinking rates among 1516 years olds between european countries and the us shows that the us is a low consumption country by european standards .
poland had a higher prevalence rate ( 65% ) of drinking in the past 30 days than the us ( 35% ) , with the highest rate being in austria ( 82% ) .
the percentage of young people in the us who reported binge drinking was lower ( 22% ) than all other european countries except turkey ( 15% ) .
screening of two polish samples ( 18 years and older ) seeking emergency services revealed males were more likely to be younger , be injured , and meet criteria for alcohol use disorders than females . among those who drank , males were more likely to report weekly drinking and 5 + drinks on an occasion than women , and nearly 50% of young women reported binge drinking .
there is also a higher incidence of reported alcohol - related mortality in men compared to women [ 4 , 13 ] , and more alcohol - related problems ( such as difficulties with marriage / partner , friends / social life , work , physical and mental health , finance , police and other authorities ) .
although few studies in poland have addressed psychosocial factors associated with alcohol misuse , one study found that a perceived imbalance in the effort - to - reward ratio at work was associated with binge drinking , increased overall alcohol intake , and problem drinking .
patterns of alcohol use among adolescents have changed as well . a study of alcohol consumption among adolescents found that rates of heavy drinking increased between 1988 and 2004 . in this study , both the type and quantity of alcohol consumed increased , reflecting shifts in the political , economic and cultural environment .
with the rapid development of hospital emergency departments ( eds ) and emergency medicine as a specialty in poland , more data are becoming available on the extent of alcohol - related injuries .
rates for all injuries ( intentional and unintentional ) in poland are higher than in the rest of the european union ( eu ) .
alcohol is one of the top ten health and injury risk factors for men and women .
a number of studies conducted among injured ed / trauma patients in poland have shown a positive association between alcohol consumption and injury [ 4 , 14 , 16 , 2931 ] . a case - crossover analysis of emergency services patients from warsaw and sosnowiec showed a four - fold risk of any injury for those drinking within 6 h compared to those who had not been drinking in both the samples .
men are more likely than women to be injured from drinking directly prior to the injury .
males who drank more than 12 l of alcohol annually were 2.4 times more likely to sustain injuries than those who drank less than 12 l annually .
injured men were twice as likely to report drinking within 6 h before the injury event than non - injured men presenting to the ed .
michalska et al . found alcohol intoxication to be a debilitating factor in prompt diagnosis , assessment , and treatment of cranio - cerebral injury .
in addition to examining overall injury rates , it is important to determine patterns of unintentional and intentional injury to be able to develop strategies to best deal with the complex interaction of alcohol and injury in the region .
unintentional injury is the fourth leading cause ( 12.8% ) of disability in males and seventh leading cause in females ( 4.8% ) in poland .
who / europe and the european commission noted that the leading causes of unintentional injury death are transportation injuries , followed by falls , poisoning , drowning , and fires .
there are very few studies conducted in poland that focus on unintentional injuries other than traffic injuries .
one study examined 1,199 patients ( 1479 years ) visiting the department of surgery at a university hospital in krakow from 19872000 for treatment of hand injuries .
of those , 320 patients ( 26.7% ) were found to be under the influence of alcohol at the time of injury , and most of them ( 89.3% ) were males and manual workers ( 74.3% ) . around 48.4% of patients suffered from injuries caused by a cut with glass , and 5.6% had operated various kinds of machines and mechanical equipment under the influence of alcohol .
most accidents happened at home ( 65.9% ) , while the others occurred in public places ( 15.9% ) , in the street ( 11.8% ) , on a farm ( 3.1% ) , and at the workplace ( 1.25% ) .
autopsies done in a study investigating the cause of mortality among polish small - scale fishermen during a 40-year period reports finding > 0.5% ( 0.5
g / l ) blood alcohol levels in 45% of deaths out of the 82 cases where autopsy was carried out ( n = 177 ) .
alcohol use was identified in 7 sea catastrophe - related cases and in 30 cases connected with other reasons for drowning with the greatest risk related to mooring , hauling , and boarding the nets .
the authors report that alcohol may be implicated in at least one - quarter of the accidents and may have contributed to other factors , such as loss of balance on deck and injury preceding falling or being swept overboard . in 2003 , 11% of all traffic accidents in poland were attributed to alcohol ; alcohol was associated with death in 13% of all traffic - related mortalities .
police reports revealed 167,318 drunk - driving cases in 2003 , out of which 18,830 of the drivers had a bac of 0.20.5 ml / g , while 148,488 had a bac of more than 0.5 ml / g .
the study at the university hospital in krakow from 19872000 reported that 7.1% ( n = 320 ) of those found to be under the influence of alcohol at the time of treatment for hand injuries were injured in traffic accidents .
there are very few studies conducted in poland that focus on unintentional injuries other than traffic injuries .
one study examined 1,199 patients ( 1479 years ) visiting the department of surgery at a university hospital in krakow from 19872000 for treatment of hand injuries .
of those , 320 patients ( 26.7% ) were found to be under the influence of alcohol at the time of injury , and most of them ( 89.3% ) were males and manual workers ( 74.3% ) . around 48.4% of patients suffered from injuries caused by a cut with glass , and 5.6% had operated various kinds of machines and mechanical equipment under the influence of alcohol .
most accidents happened at home ( 65.9% ) , while the others occurred in public places ( 15.9% ) , in the street ( 11.8% ) , on a farm ( 3.1% ) , and at the workplace ( 1.25% ) .
autopsies done in a study investigating the cause of mortality among polish small - scale fishermen during a 40-year period reports finding > 0.5% ( 0.5
g / l ) blood alcohol levels in 45% of deaths out of the 82 cases where autopsy was carried out ( n = 177 ) .
alcohol use was identified in 7 sea catastrophe - related cases and in 30 cases connected with other reasons for drowning with the greatest risk related to mooring , hauling , and boarding the nets .
the authors report that alcohol may be implicated in at least one - quarter of the accidents and may have contributed to other factors , such as loss of balance on deck and injury preceding falling or being swept overboard .
in 2003 , 11% of all traffic accidents in poland were attributed to alcohol ; alcohol was associated with death in 13% of all traffic - related mortalities .
police reports revealed 167,318 drunk - driving cases in 2003 , out of which 18,830 of the drivers had a bac of 0.20.5 ml / g , while 148,488 had a bac of more than 0.5 ml / g .
the study at the university hospital in krakow from 19872000 reported that 7.1% ( n = 320 ) of those found to be under the influence of alcohol at the time of treatment for hand injuries were injured in traffic accidents .
the rates of homicide in poland are higher than in the rest of the eu .
intentional injury is also the sixth leading cause ( 4.8% ) of disability in males .
studies conducted specifically in poland , as well as those conducted across several other countries , have shown that alcohol consumption often precedes violent events [ 30 , 3841 ] . in samples of ed patients from warsaw and sosnowiec , poland , cherpitel et al .
( 2005 ) found that there was over a 17-fold increased risk for a violence - related injury for individuals who reported drinking 6 h before the injury , the risk being greater for women than for men .
an epidemiological investigation of the prevalence of potentially traumatic events ( ptes ) in a sample of alcohol - dependent patients in poland found that the numbers of ptes reported were related to higher post - traumatic stress , but not necessarily to higher alcohol use intensity . a higher level of alcohol misuse was associated with two categories of ptes : robbery and mugging , and witnessing death or assault .
alcohol has been found to be a strong risk factor for intimate partner violence in poland .
makara - studzinska et al . analyzed data from a survey of 800 participants ( 400 perpetrators and their 400 victims of domestic violence ) in the office of the association for violence prevention in the city of lublin , poland .
results suggested that perpetrators with a history of alcohol abuse were more likely to be younger , had less education , were less likely to be permanently employed , and had more problems with the legal system than perpetrators without a history of alcohol abuse . in the same sample , 76% of domestic violence perpetrators had problems related to alcohol , as measured by an audit score > 8 .
in addition , another study in the country found 25% of surveyed pregnant women reported physical and emotional abuse by their partners .
men that physically abused pregnant women in this study were more often found to have primary school education , be unemployed , and drink alcohol .
a substantial number of 15 to 20 year olds in poland reported having experienced at least one of four kinds of parent - child violence ( emotional , physical , negligence , and sexual abuse ) , of which parental alcohol abuse was one of the most frequently cited reasons for family violence .
in poland , suicide is the leading cause of intentional injury - related death , and suicide rates are higher than in the rest of the eu . from 1989 to 1994 , a time of rapid social and political change , the suicide rate in poland increased by 26.5% .
the study aimed at describing suicide mortality in eastern europe by investigating the covariation between changes in some of the hypothesized causes such as alcohol consumption , economic situation , general social stress , political situation , societal ( dis)organization , and the changes in the suicide rates . the linear regression model , which consisted of variables such as hypothetical general stress ( as indicated by mortality / life expectancy ) , democratization , alcohol consumption , and social disorganization ( with a period - dependent effect ) , was able to predict percentual changes in the suicide rates in 16 out of the 28 eastern bloc countries from 19841989 and 19891994 fairly accurately , but failed to do so for poland besides other countries .
on the other hand , landberg ( 2008 ) noted that the relationship between alcohol consumption and suicide mortality tends to be strongest in countries with detrimental drinking patterns , such as poland ( based on who s hazardous patterns score ) . in this cross - cultural study that included poland , a 1-l increase in per capita consumption of alcohol
was associated with an increase in overall suicide rates by 5.77.5% , with males at higher risk than females . in another study , among 154 alcohol - dependent patients admitted to addiction treatment centers in poland , 43% reported attempting suicide at some point during their lifetime .
post - mortem reports have shown the presence of alcohol in almost 50% of polish suicide victims .
given the strong association between alcohol consumption and injury in poland , it is imperative that medical professionals be prepared to respond .
academic emergency departments , the polish emergency medicine society , and psychology and social service societies , among other entities , will be key in the development and delivery of training materials to emergency , primary care , social service , and addiction treatment professionals . based on training recommendations for the us and other countries , training of current and future ed physicians , nurses , emts , social workers , and psychologists in poland and eastern europe in general should include the following areas :
recognition and assessmentto ensure that clinicians are knowledgeable in assessing the quantity and frequency of alcohol and other drug use among patients seen in medical and social service settings , key recommendations include : ( 1 ) increasing preclinical and clinical training regarding ( a ) the available alcohol screening instruments ; ( b ) methods to determine other medication and drug use [ including over - the - counter ( otc ) medications and herbal agents ] ; ( c ) techniques for conducting screening ; ( d ) techniques to incorporate structured screening for alcohol , medications , and illicit drugs into standard emergency practice ; and ( 2 ) developing preclinical and clinical training evaluations and tests that include questions on substance use screening.a study comparing performance of alcohol screening instruments to identify alcohol use disorders in the emergency services setting in the warsaw and sosnowiec regions of poland found sensitivity of the raps4 and alcohol use disorders identification test ( audit ) to be significantly better than the cage for alcohol dependence among males in warsaw , but specificity to be poorer in males in both the samples .
the cage appeared to perform considerably better for both males and females in sosnowiec than in warsaw . among females ,
sensitivity for alcohol abuse / harmful drinking and for alcohol dependence or abuse / harmful drinking was significantly better for the raps4-qf than for the cage or audit at a cutoff point of 8 across both sites .
however , a smaller - scale research study found that the audit questionnaire was more sensitive than the cage when implemented in polish emergency departments .
this finding is supported by a number of studies in the us and uk in a variety of medical settings [ 5053 ] .
the audit has been found to have good psychometric properties in a number of countries with a variety of patient populations.conducting structured and targeted brief advice / interventionsadvice and brief interventions can be delivered as part of early detection programs in eds and other medical and psychosocial treatment settings .
brief alcohol interventions such as tailored or generic booklets with brief counseling or motivational interviewing with an interventionist have been proven to be effective in a variety of medical settings , including the emergency department [ 5459 ] .
brief interventions have two purposes : ( 1 ) to help individuals who are classified as at - risk for alcohol problems to cut back or stop using alcohol or drugs , and ( 2 ) to help individuals with more serious substance use problems engage in needed treatment .
educational materials are available through the national institute on alcohol abuse and alcoholism ( niaaa ) and through the center for substance abuse treatment ( csat ) in the treatment improvement protocol ( tip ) # 34 ,
brief interventions and brief therapies for substance abuse , as well as through the sbirt ( screening , brief intervention , referral , and treatment ) quick guide .training can be made available to professionals who work in emergency care , primary care , and psychosocial programs through preclinical and clinical education programs , continuing education credits , and other workshop opportunities .
these are needed because , in a changing health care delivery system , brief , effective methods to deal with alcohol problems will be both clinically and cost effective .
one such short , concentrated training session at yale university ed in connecticut significantly improved knowledge and practice of screening and interventions for alcohol problems among a sample of emergency medicine residents , while the control group at harvard university in massachusetts showed no changes .
there are very few preclinical and clinical programs that currently include any of the techniques of brief alcohol interventions in poland.because the ed is a very fast - paced , clinically demanding venue , training personnel in how to identify and manage patients with at - risk and problem drinking should also be accompanied by research on methods to best implement some of the newer , innovative technologies for screening and intervening .
the use of computerized tailored interventions represents one such technique to provide targeted , individualized feedback to injured and medical ed patients .
other technologies that may be useful in the future in poland include the use of interactive voice recognition ( ivr ) technology to facilitate screening , delivery of educational interventions , and follow - up on patient progress over the telephone .
interactive computer programs on laptops or palmtops ; web - based interventions ; computerized bundling of brief health messages for multiple health risks ( e.g. , smoking , alcohol use , seat belt use ) ; and audio interventions tailored to specific problems and delivered through headsets are all potential approaches in emergency , urgent care , and primary care settings .referral to treatmentalso , providers need to have knowledge of the available in- and outpatient programs to help those individuals who meet criteria for more serious substance use problems .
the brief intervention structure provides the opportunity to work with patients who have more serious alcohol problems and to help them consider obtaining formal substance abuse treatment.it is ideal to have psychiatric emergency services co - located in the ed with acute medical and injury care whenever possible to address patients who screen positive for risky alcohol use . alternatively increased collaboration and consultation between ed and mental health services is critical to any successful screening and brief intervention program
. however , specialty resources are limited and often can not be provided during the acute care time frame .
the training recommendations provided are focused on the many eds and clinicians that do not have access or availability to provide this service by specialty psychiatric services .
recognition and assessment to ensure that clinicians are knowledgeable in assessing the quantity and frequency of alcohol and other drug use among patients seen in medical and social service settings , key recommendations include : ( 1 ) increasing preclinical and clinical training regarding ( a ) the available alcohol screening instruments ; ( b ) methods to determine other medication and drug use [ including over - the - counter ( otc ) medications and herbal agents ] ; ( c ) techniques for conducting screening ; ( d ) techniques to incorporate structured screening for alcohol , medications , and illicit drugs into standard emergency practice ; and ( 2 ) developing preclinical and clinical training evaluations and tests that include questions on substance use screening .
a study comparing performance of alcohol screening instruments to identify alcohol use disorders in the emergency services setting in the warsaw and sosnowiec regions of poland found sensitivity of the raps4 and alcohol use disorders identification test ( audit ) to be significantly better than the cage for alcohol dependence among males in warsaw , but specificity to be poorer in males in both the samples .
the cage appeared to perform considerably better for both males and females in sosnowiec than in warsaw . among females ,
sensitivity for alcohol abuse / harmful drinking and for alcohol dependence or abuse / harmful drinking was significantly better for the raps4-qf than for the cage or audit at a cutoff point of 8 across both sites .
however , a smaller - scale research study found that the audit questionnaire was more sensitive than the cage when implemented in polish emergency departments .
this finding is supported by a number of studies in the us and uk in a variety of medical settings [ 5053 ] .
the audit has been found to have good psychometric properties in a number of countries with a variety of patient populations . conducting structured and targeted brief advice / interventions advice and brief interventions
can be delivered as part of early detection programs in eds and other medical and psychosocial treatment settings .
brief alcohol interventions such as tailored or generic booklets with brief counseling or motivational interviewing with an interventionist have been proven to be effective in a variety of medical settings , including the emergency department [ 5459 ] .
brief interventions have two purposes : ( 1 ) to help individuals who are classified as at - risk for alcohol problems to cut back or stop using alcohol or drugs , and ( 2 ) to help individuals with more serious substance use problems engage in needed treatment .
educational materials are available through the national institute on alcohol abuse and alcoholism ( niaaa ) and through the center for substance abuse treatment ( csat ) in the treatment improvement protocol ( tip ) # 34 ,
brief interventions and brief therapies for substance abuse , as well as through the sbirt ( screening , brief intervention , referral , and treatment ) quick guide .
training can be made available to professionals who work in emergency care , primary care , and psychosocial programs through preclinical and clinical education programs , continuing education credits , and other workshop opportunities .
these are needed because , in a changing health care delivery system , brief , effective methods to deal with alcohol problems will be both clinically and cost effective .
one such short , concentrated training session at yale university ed in connecticut significantly improved knowledge and practice of screening and interventions for alcohol problems among a sample of emergency medicine residents , while the control group at harvard university in massachusetts showed no changes .
there are very few preclinical and clinical programs that currently include any of the techniques of brief alcohol interventions in poland . because the ed is a
very fast - paced , clinically demanding venue , training personnel in how to identify and manage patients with at - risk and problem drinking should also be accompanied by research on methods to best implement some of the newer , innovative technologies for screening and intervening .
the use of computerized tailored interventions represents one such technique to provide targeted , individualized feedback to injured and medical ed patients .
other technologies that may be useful in the future in poland include the use of interactive voice recognition ( ivr ) technology to facilitate screening , delivery of educational interventions , and follow - up on patient progress over the telephone .
interactive computer programs on laptops or palmtops ; web - based interventions ; computerized bundling of brief health messages for multiple health risks ( e.g. , smoking , alcohol use , seat belt use ) ; and audio interventions tailored to specific problems and delivered through headsets are all potential approaches in emergency , urgent care , and primary care settings .
referral to treatment also , providers need to have knowledge of the available in- and outpatient programs to help those individuals who meet criteria for more serious substance use problems .
the brief intervention structure provides the opportunity to work with patients who have more serious alcohol problems and to help them consider obtaining formal substance abuse treatment .
it is ideal to have psychiatric emergency services co - located in the ed with acute medical and injury care whenever possible to address patients who screen positive for risky alcohol use .
alternatively increased collaboration and consultation between ed and mental health services is critical to any successful screening and brief intervention program . however , specialty resources are limited and often can not be provided during the acute care time frame .
the training recommendations provided are focused on the many eds and clinicians that do not have access or availability to provide this service by specialty psychiatric services .
the burden of alcohol - related injury will likely increase as poland s economic and social structure continue to change resulting from admission to the eu , changes in the employment structure , and coping with a global recession .
emergency departments need to be prepared to implement alcohol screening and intervention practices in order to effectively target and treat patients with substance use disorders and prevent future alcohol - associated re - injury , readmissions , and mortality .
the health care delivery system in poland must adopt new and innovative methods that work with a range of patient populations who have drinking patterns that put them at risk for intentional and unintentional injuries , problems related to physical and mental health functioning , employment , and social / family problems .
alcohol use and misuse in poland , and eastern europe more generally , and the resulting burden of injuries have made it critically important to address this important public health concern by developing surveillance and prevention strategies to limit future morbidity and mortality .
knowledge gained from addressing alcohol problems in this strategically important region of the world can have wide - ranging implications for global health interventions and policies with regards to alcohol problems . | backgroundalcohol plays a significant role in accidents , injuries , and their outcomes . according to the world health organization ( who ) , there are 76.3 million people with alcohol use disorders worldwide ;
in 2000 , 1.8 million deaths and loss of 58.3 million disability - adjusted life years were attributed to alcohol.methodsalthough the association between alcohol consumption and trauma - related morbidity and mortality is well - documented , particularly in the us , there is much less information on trauma and alcohol in poland , a country undergoing unprecedented economic and cultural changes stemming from entry into the european union ( eu ) in the midst of a global recession.resultsinjury is the third leading cause of death in poland .
rates for all injuries in poland are higher than in the rest of the eu .
alcohol is one of the greatest risk factors for disease and injury among men and one of the top ten health and injury risk factors for women . in this paper
we review the last 10 years of research on injury and alcohol in poland.conclusionrecommendations are provided for next steps with regard to training health care professionals in emergency medical settings to address this serious and growing problem . |
in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs .
health and social services today face groups of patients who have composite problems and are often unstable .
they include , very obviously , elderly persons with multiple problems , chronically ill children , and persons suffering mental ill - health .
they have continuing need of care and in search of care they move between primary care , hospital care and municipality care , such as that provided for elderly persons .
their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected .
individuals do not see themselves as multi - ill , but as needing support for their needs as they know them .
it must be said that from the 1970s onwards a number of integrative approaches have been tried out , not least in education .
although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs .
so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives .
much of the evidence indicates that the problem we face is a result of the prevailing mindset .
how can we increase our understanding of health and social services that are located in different organizations ?
in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs .
health and social services today face groups of patients who have composite problems and are often unstable .
they include , very obviously , elderly persons with multiple problems , chronically ill children , and persons suffering mental ill - health .
they have continuing need of care and in search of care they move between primary care , hospital care and municipality care , such as that provided for elderly persons .
their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected .
individuals do not see themselves as multi - ill , but as needing support for their needs as they know them .
it must be said that from the 1970s onwards a number of integrative approaches have been tried out , not least in education .
although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs .
so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives .
much of the evidence indicates that the problem we face is a result of the prevailing mindset .
how can we increase our understanding of health and social services that are located in different organizations ?
lindberg observed examples of meaningful co - operation at the local level , with colleagues from different organizations meeting and pooling their knowledge of local conditions with the patient or user as the focal point .
the phenomenon has been variously called the chain of care , integrated care , seamless care or shared care .
this co - operation aims at creating a continuing relationship with the patient / user regardless of who at a given moment is the responsible provider .
edgren and stenberg found these practical attributes of co - operation in cass :
a common task is shared among the co - working agents.collaboration is in people s minds , it is instinctive behaviour.each actor s capabilities are known and respected.a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions .
collaboration is in people s minds , it is instinctive behaviour . each actor s capabilities are known and respected .
a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions . according to brommels in his presentation to the ehma - conference 2006
, the cas approach means , among other things :
identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts.avoiding strict definitions of roles and concentrating on agreed actions.giving agents their freedom to organize current activities .
identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts . avoiding strict definitions of roles and concentrating on agreed actions . giving agents their freedom to organize current activities .
if we are to improve our understanding of how a health or social service provider functions as an integrated part of a locally driven health and social service system , we need an innovative , in terms of changing practice , model to guide our thinking . traditional models view systems as machines .
it helps us understand what happens in dynamic living systems , where many agents are interconnected . in order to make clear its significance let us first recapitulate the machine model . for a long time effective organizations
the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate .
hospitals and other health services organizations are usually designed to function according to principles of scientific management [ 15 , 16 ] .
rationality , objectivity , stability and predictability are the terms we associate with this approach .
rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system .
change is seen as a linier and predictable process , controlled by top managers and carried out by works managers .
plans are made and are to be followed , and the intended change , takes place as a direct consequence . if problems do arise during the implementation of change , then either there is wrong expectations or there is an inability or refusal to take prescribed action .
the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called
and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this
we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened .
if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . instead of theories assuming cause and effect linkages between separate details , we need theories to deal with patterns and principles .
a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown .
davis coined the expression any time , any place when he describes how time and space restrictions
this has given us enormous possibilities to communicate , to become connected , to network .
as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . indeed , most of our assumptions about organizations are at least fifty years old .
so why turn to complexity science and to complex adaptive systems ( cas ) ? according to zimmerman et al .
they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool .
it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care .
complexity can be expressed as the amount of information needed to describe or understand something . and
the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system .
a cas consists of several subsystems called agents , which act in dependence of one another .
. they may either compete or co - operate according to their sense of their interests and what will bring them an advantage .
self - organization is about creating order or increasing the regularity of the system without help from the outside .
good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team .
important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere .
researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically .
prominent figures in the field include nobel laureates ilya prigogine in chemistry [ 26 , 27 ] and murray gell - mann in theoretical physics . within medicine
we find stuart kauffman [ 30 , 31 ] and physics russ marion and mary uhl - bien .
according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task .
the more a task is characterized by regularities the more we can think in terms of applying routines .
when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity .
although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services .
the agents see the point , because they create order out of many local interactions .
complexity science offers new ways to understand how complementary knowledge organizes for co - operation .
we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system .
emergency treatment is a clear example of independent agents interacting locally with other independent agents .
another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants .
the objective was to identify how local politicians and managers understand problems and goals regarding the structures and processes involved in the care of the elderly . a common vision , easy to understand and to communicate ,
there is no external constructor , no superior centrally located source to govern the design of the system .
when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today
digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and
thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration .
complexity science emphasizes the inherent power of development and self - organizing nature of the system .
attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive .
in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient .
such top down attempts are usually made in order to make it simpler for the provider not the person seeking care .
when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them .
one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation .
later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other .
so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace .
positive feedback loops enhance or amplify changes and tend to move a system away from equilibrium stage .
for example in general terms , there is a positive feedback loop between income and consumption in an economy . the bigger the income of the individuals ,
negative feedbacks tend to dampen or buffer changes and hold a system to some equilibrium state , like a thermostat in a fridge . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science positive and negative feedback
there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives
. it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours .
the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . with their local knowledge of needs ,
they are so much better placed to act than any centrally located management ever could be .
they sense that they have control over their own work situation , perhaps the most important change needed to create the good workplace . in this way
we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole .
general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas .
others may argue that this is only one of several possible approaches to promote integrated care .
communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted .
a certain level of system inertia may also develop over time . to learn to use a different approach to organizing may mean insecurity and risk similar to being expected to accept new technology .
seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management .
the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria .
first this is due to the nature of the system which is determined by the sum of choices made in it .
secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! we know that all our choices to some extent represent a step in the dark , and therefore , we can not but be responsible for them .
but the cas approach lays out the considerations to be taken into account more clearly than the alternatives .
for a long time effective organizations have been looked upon like well - oiled machines .
the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate .
hospitals and other health services organizations are usually designed to function according to principles of scientific management [ 15 , 16 ] .
rationality , objectivity , stability and predictability are the terms we associate with this approach .
rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system .
change is seen as a linier and predictable process , controlled by top managers and carried out by works managers .
plans are made and are to be followed , and the intended change , takes place as a direct consequence . if problems do arise during the implementation of change , then either there is wrong expectations or there is an inability or refusal to take prescribed action .
the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called
and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this
we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened .
if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . instead of theories assuming cause and effect linkages between separate details , we need theories to deal with patterns and principles .
a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown .
davis coined the expression any time , any place when he describes how time and space restrictions
this has given us enormous possibilities to communicate , to become connected , to network .
as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . indeed , most of our assumptions about organizations are at least fifty years old .
so why turn to complexity science and to complex adaptive systems ( cas ) ? according to zimmerman et al .
they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool .
it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care .
complexity can be expressed as the amount of information needed to describe or understand something . and an important part of complexity science is the complex adaptive system ( cas ) .
the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system .
a cas consists of several subsystems called agents , which act in dependence of one another .
. they may either compete or co - operate according to their sense of their interests and what will bring them an advantage .
self - organization is about creating order or increasing the regularity of the system without help from the outside .
good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team .
important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere .
researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically .
prominent figures in the field include nobel laureates ilya prigogine in chemistry [ 26 , 27 ] and murray gell - mann in theoretical physics . within medicine
we find stuart kauffman [ 30 , 31 ] and physics russ marion and mary uhl - bien .
according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task .
the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity .
although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services .
the agents see the point , because they create order out of many local interactions .
complexity science offers new ways to understand how complementary knowledge organizes for co - operation .
we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system .
emergency treatment is a clear example of independent agents interacting locally with other independent agents .
another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants .
the objective was to identify how local politicians and managers understand problems and goals regarding the structures and processes involved in the care of the elderly .
a common vision , easy to understand and to communicate , was created to connect the two groups .
there is no external constructor , no superior centrally located source to govern the design of the system .
when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today
digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration .
complexity science emphasizes the inherent power of development and self - organizing nature of the system .
attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive . in practice
we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient .
such top down attempts are usually made in order to make it simpler for the provider not the person seeking care . working in isolation the burden of coordination passes to the patient .
when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them .
one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation .
later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other .
so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace .
positive feedback loops enhance or amplify changes and tend to move a system away from equilibrium stage .
for example in general terms , there is a positive feedback loop between income and consumption in an economy .
the bigger the income of the individuals , the more the whole population consume , which further increasing their income as individuals .
negative feedbacks tend to dampen or buffer changes and hold a system to some equilibrium state , like a thermostat in a fridge . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science
there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives
it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours .
the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . with their local knowledge of needs ,
they are so much better placed to act than any centrally located management ever could be .
they sense that they have control over their own work situation , perhaps the most important change needed to create the good workplace . in this way
we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole .
general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . some theorists would claim that cas is nothing but the emperor s new clothes .
others may argue that this is only one of several possible approaches to promote integrated care .
communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted .
a certain level of system inertia may also develop over time . to learn to use a different approach to organizing may mean insecurity and risk similar to being expected to accept new technology .
seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management .
the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria .
first this is due to the nature of the system which is determined by the sum of choices made in it .
secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! we know that all our choices to some extent represent a step in the dark , and therefore , we can not but be responsible for them .
but the cas approach lays out the considerations to be taken into account more clearly than the alternatives .
when the competence necessary for carrying out a given task does not lie within one individual provider organization , co - operation between agents within the system comes into play to discharge that task .
we are no longer talking about the individual organization / agent but shaping overall workable solutions taking a patient / user perspective .
this applies to matching care with the needs of different patient groups and of individual patients .
the cas approach helps the management to understand why the traditional top down way of managing may meet with problems in organizations with complex tasks .
an important discussion is about how the top management in fact executes its steering function .
leaders may consider accepting complexity instead of trying to reduce it , formulate few simple and concrete goals , communicate and give feedback and measure performance .
when we perceive health and social service organizations as cass we should gain more insight into the processes that go on within and between organizations .
are we willing to face the interdependence between health and social services , the dependence on collaboration to deliver appropriate integrated care ?
if we do , complexity science could be an important step towards fresh thinking in order to fulfil our patients and users presently unfulfilled needs .
edgar borgenhammar , phd ( economics ) , mba ( berkeley ) , dsi ( lund ) , professor emeritus , from the nordic school of public health in gteborg , former hospital chief executive , sweden peter carswell , dr . , centre for health services research and policy , faculty of medical and health sciences , university of auckland , auckland , new zealand alene hokenstad , project director , united hospital fund , new york , usa | introductionorganizations can be regarded as systems .
the traditional model of systems views them as machines .
this seems to be insufficient when it comes to understanding and organizing complex tasks . to better understand integrated care we should approach organizations as constantly changing living organisms , where many agents are interconnected in so - called complex adaptive systems ( cas).theory and discussionthe term complex
emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system .
adaptive means that system change occurs through successive adaptations .
a cas consists of several subsystems called agents , which act in dependence of one another .
examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . studying a cas ,
the focus is on the interaction and communication between agents .
although these thoughts are not new , the cas - approach has not yet been widely applied to the management of integrated care .
this helps the management to understand why the traditional top down way of managing , following the machine model thinking , may meet with problems in interdependent organizations with complex tasks.conclusionwhen we perceive health and social services as cass we should gain more insight into the processes that go on within and between organizations and how top management , for example within a hospital , in fact executes its steering function . |
carfilzomib ( cfz ) , an epoxyketone with specific chymotrypsin - like activity , is a second - generation proteasome inhibitor with significant activity in patients with relapsed and refractory multiple myeloma . on july 20 , 2012 , the us food and drug administration approved cfz to treat patients with multiple myeloma who have received at least two prior therapies including bortezomib ( bort ) and an immunomodulatory agent and have demonstrated disease progression on or within 60 days of completion of the last therapy .
cytogenetic abnormalities did not appear to have a significant impact on the cfz activity .
carfilzomib was well tolerated and demonstrated promising efficacy in patients with renal insufficiency .
pomalidomide ( pom ) ( cc-4047 ) is a novel immunomodulatory derivative ( imid ) with a stronger in vitro anti - myeloma effect compared with older imids thalidomide and lenalidomide ( len ) . on february 8 , 2013 ,
the us food and drug administration approved pom ( pomalyst , celgene ) for the treatment of mm patients who have received at least two prior therapies including len and bort and have demonstrated progression on or within 60 days of completion of the last therapy .
pomalidomide is a novel imid with significant anti - myeloma activity and manageable toxicity .
this compound has shown high efficacy in mm patients who were resistant to prior use of len / bort as well as in patients with a high - risk cytogenetic profile .
carfilzomib and pom have very high efficacy and will be used also in first line therapy in future . |
|
guillain - barr syndrome ( gbs ) is the most common cause of acute neuromuscular paralysis .
the most frequent variants of gbs include acute inflammatory demyelinating polyradiculoneuropathy ( aidp ) and acute motor axonal neuropathy ( aman ) .
campylobacter jejuni infection precedes the onset of gbs in 26% of the cases in western hemisphere and in two - thirds of patients in china , and it seems to be exclusively associated with aman .
the aman variant of gbs is most frequently associated with antibodies to gangliosides gm1 , gd1a , and galnac - gd1a .
the association of gbs with antecedent c. jejuni infection proposed the mechanism of molecular mimicry in the immunopathogenesis of the disease .
a specific serotype of penner 's 19 ( o:19 ) is much more frequently isolated from gbs patients than from enteritis patients .
the role of antibodies to the peripheral nerve myelin proteins and glycoproteins was not sufficiently investigated in gbs .
gm1-positive sera from patients with gbs following infection with c. jejuni showed reactivity to a 63-kda flagellar protein purified from c. jejuni ( o:19 ) .
it has also been shown that gm1 antibodies cross - react with gal - galnac - bearing glycoproteins from the peripheral nerve .
we present the results of cross - reactivity of gm1-positive serum with several gal - galnac - bearing glycoproteins isolated from the human peripheral nerve and from c. jejuni ( o:19 ) , including the glycoprotein with electrophoretic mobility between 60 and 70 kda , present in both isolates .
these data indicated the possible role of some protein antigens from c. jejuni in the pathogenesis of gbs .
determination of the molecular structure of the glycoprotein structures present in the human peripheral nerve and the bacteria c. jejuni is necessary for elucidation of their antigenicity .
the aim of this study was to examine the reactivity of the peptides obtained after digestion with trypsin of the cross - reactive glycoproteins isolated from the human peripheral nerve and c. jejuni ( o:19 ) with peanut agglutinin ( pna ) as a marker for the gal - galnac determinant and with sera from patients with gbs .
human peripheral nerve was obtained at autopsy within 8 h after death from patients who died from non - neurological disease ; it was kept frozen at -70 c ( department of forensic medicine , faculty of medicine , university ss .
the neural tissue was pulverized in liquid nitrogen , delipidated with chloroform : methanol ( 1:2 ) solution , solubilized by homogenization in 0.5% triton x-100 , 0.4% sodium dodecyl sulfate ( sds ) with protease inhibitor cocktail , and heated at 65c for 10 min .
the insoluble matter was removed by centrifugation at 4,200 rpm for 45 min at room temperature . the c. jejuni serotype ( o:19 ) ( obtained from atcc 43446 )
the identity of c. jejuni was confirmed by microscopic examination , by determining the mobility , staining according to gram and with biochemical tests at the institute for microbiology and parasitology , faculty of medicine , skopje .
bacterial cells were collected in 0.9% nacl and centrifuged at 4,000 rpm for 30 min .
pellets were resuspended in 8.0 ml of 0.1 m tris - hcl ( ph 7.8 ) and disrupted by a ultrasonic cell disruptor ( microson , ultrasonic cell disruptor xl , misonix incorporated , new york , usa ) .
after centrifugation ( 45 min , 4,200 rpm ) , the proteins in the supernatant were dialyzed twice against 0.1 m tris - hcl ( ph 7.5 ) at 4c for 3 h. gal - galnac - bearing glycoproteins from the human peripheral nerve and c. jejuni ( o:19 ) were purified by affinity chromatography , using agarose - bound pna .
purified glycoproteins isolated from the peripheral nerve and c. jejuni ( o:19 ) were separated on 7.5% acrylamide / bisacrylamide gel ( 20 g total glycoproteins per well , 1 g purified glycoprotein per well ) by sds - polyacrylamide gel electrophoresis ( sds - page ) and transferred electrophoretically onto nitrocellulose sheets .
unreactive binding sites were blocked in 8% bovine serum albumin ( bsa ) in tris - buffered saline ( tbs - 0.02 m tris base , 0.5 m nacl , ph 7.5 ) for 1 hour at room temperature .
the blots were washed three times with tbs containing 1% tween 20 and incubated overnight at 4c with biotinylated pna ( sigma - aldrich ) ( 10 g / ml ) and with sera from 10 patients with gbs and high titer of anti - gm1 antibodies ( institute of neurology , clinical center of serbia , belgrade , serbia ) diluted 1:100 .
after washing , the strips were incubated with avidin conjugated with peroxidase ( 1:1000 ) , and with anti - human igg antibodies conjugated with peroxidase ( 1:500 ) for 1 hour at room temperature .
visualization of the reaction products was done using diaminobenzidine ( dab ) , 15 mm imidazole , and 0.025% h2o2 in water , ( biorad laboratories , hercules , ca , usa ) .
gal - galnac bearing glycoproteins were electrophoretically separated using 7.5% acrylamide / bisacrylamide gel ( approximately 20 g total glycoproteins / well ) .
after electrophoresis , a strip from the left side of the gel , including molecular weight marker and first line of the protein sample , was cut with a razor and stained with 0.1% amido black , 1% acetic acid , and 40% methanol in water .
this strip was used as a reference gel strip to localize the bands of glycoproteins with mobility between 60 and 70 kda . after aligning the reference strip with the unstained gel portion
, the bands of the gel that align with the stained protein of interest were cut from the unstained gel and rinsed with distilled water .
gels containing proteins of interest were equilibrated in 1 ml 0.125 m tris ( ph 6.8 ) for 10 - 15 min and added to the wells of a 15% acrylamide / bisacrylamide gel .
enzyme trypsin in buffer was added to the wells and after 10 - 15 min electrophoresis was run .
after the electrophoretic separation , the gel was stained with silver stain ( silver stain plus , biorad laboratories , hercules , ca , usa ) for visualization of the peptides .
unreactive binding sites were blocked in 8% bsa in tbs ( 0.02 m tris base , 0.5 m nacl , ph 7.5 ) for 1 hour at room temperature .
the blots were washed three times with tbs containing 1% tween 20 and incubated overnight at 4c with biotinylated pna ( sigma - aldrich ) ( 10 g / ml ) and with the sera from patients with gbs ( 1:100 ) .
after washing , the strips were incubated with avidin conjugated with peroxidase ( 1:1000 ) , and with anti - human igg antibodies conjugated with peroxidase ( 1:500 ) for 1 hour at room temperature .
visualization of the reaction products was done using dab , 15 mm imidazole , and 0.025% h2o2 in water ( biorad laboratories , hercules , ca , usa ) .
human peripheral nerve was obtained at autopsy within 8 h after death from patients who died from non - neurological disease ; it was kept frozen at -70 c ( department of forensic medicine , faculty of medicine , university ss .
the neural tissue was pulverized in liquid nitrogen , delipidated with chloroform : methanol ( 1:2 ) solution , solubilized by homogenization in 0.5% triton x-100 , 0.4% sodium dodecyl sulfate ( sds ) with protease inhibitor cocktail , and heated at 65c for 10 min .
the insoluble matter was removed by centrifugation at 4,200 rpm for 45 min at room temperature . the c. jejuni serotype ( o:19 ) ( obtained from atcc 43446 )
the identity of c. jejuni was confirmed by microscopic examination , by determining the mobility , staining according to gram and with biochemical tests at the institute for microbiology and parasitology , faculty of medicine , skopje .
bacterial cells were collected in 0.9% nacl and centrifuged at 4,000 rpm for 30 min .
pellets were resuspended in 8.0 ml of 0.1 m tris - hcl ( ph 7.8 ) and disrupted by a ultrasonic cell disruptor ( microson , ultrasonic cell disruptor xl , misonix incorporated , new york , usa ) .
after centrifugation ( 45 min , 4,200 rpm ) , the proteins in the supernatant were dialyzed twice against 0.1 m tris - hcl ( ph 7.5 ) at 4c for 3 h.
gal - galnac - bearing glycoproteins from the human peripheral nerve and c. jejuni ( o:19 ) were purified by affinity chromatography , using agarose - bound pna .
purified glycoproteins isolated from the peripheral nerve and c. jejuni ( o:19 ) were separated on 7.5% acrylamide / bisacrylamide gel ( 20 g total glycoproteins per well , 1 g purified glycoprotein per well ) by sds - polyacrylamide gel electrophoresis ( sds - page ) and transferred electrophoretically onto nitrocellulose sheets .
unreactive binding sites were blocked in 8% bovine serum albumin ( bsa ) in tris - buffered saline ( tbs - 0.02 m tris base , 0.5 m nacl , ph 7.5 ) for 1 hour at room temperature .
the blots were washed three times with tbs containing 1% tween 20 and incubated overnight at 4c with biotinylated pna ( sigma - aldrich ) ( 10 g / ml ) and with sera from 10 patients with gbs and high titer of anti - gm1 antibodies ( institute of neurology , clinical center of serbia , belgrade , serbia ) diluted 1:100 .
after washing , the strips were incubated with avidin conjugated with peroxidase ( 1:1000 ) , and with anti - human igg antibodies conjugated with peroxidase ( 1:500 ) for 1 hour at room temperature .
visualization of the reaction products was done using diaminobenzidine ( dab ) , 15 mm imidazole , and 0.025% h2o2 in water , ( biorad laboratories , hercules , ca , usa ) .
purified glycoproteins isolated from the peripheral nerve and c. jejuni ( o:19 ) were separated on 7.5% acrylamide / bisacrylamide gel ( 20 g total glycoproteins per well , 1 g purified glycoprotein per well ) by sds - polyacrylamide gel electrophoresis ( sds - page ) and transferred electrophoretically onto nitrocellulose sheets .
unreactive binding sites were blocked in 8% bovine serum albumin ( bsa ) in tris - buffered saline ( tbs - 0.02 m tris base , 0.5 m nacl , ph 7.5 ) for 1 hour at room temperature .
the blots were washed three times with tbs containing 1% tween 20 and incubated overnight at 4c with biotinylated pna ( sigma - aldrich )
( 10 g / ml ) and with sera from 10 patients with gbs and high titer of anti - gm1 antibodies ( institute of neurology , clinical center of serbia , belgrade , serbia ) diluted 1:100 .
after washing , the strips were incubated with avidin conjugated with peroxidase ( 1:1000 ) , and with anti - human igg antibodies conjugated with peroxidase ( 1:500 ) for 1 hour at room temperature .
visualization of the reaction products was done using diaminobenzidine ( dab ) , 15 mm imidazole , and 0.025% h2o2 in water , ( biorad laboratories , hercules , ca , usa ) .
gal - galnac bearing glycoproteins were electrophoretically separated using 7.5% acrylamide / bisacrylamide gel ( approximately 20 g total glycoproteins / well ) .
after electrophoresis , a strip from the left side of the gel , including molecular weight marker and first line of the protein sample , was cut with a razor and stained with 0.1% amido black , 1% acetic acid , and 40% methanol in water .
this strip was used as a reference gel strip to localize the bands of glycoproteins with mobility between 60 and 70 kda . after aligning the reference strip with the unstained gel portion
, the bands of the gel that align with the stained protein of interest were cut from the unstained gel and rinsed with distilled water .
gels containing proteins of interest were equilibrated in 1 ml 0.125 m tris ( ph 6.8 ) for 10 - 15 min and added to the wells of a 15% acrylamide / bisacrylamide gel .
enzyme trypsin in buffer was added to the wells and after 10 - 15 min electrophoresis was run .
after the electrophoretic separation , the gel was stained with silver stain ( silver stain plus , biorad laboratories , hercules , ca , usa ) for visualization of the peptides .
unreactive binding sites were blocked in 8% bsa in tbs ( 0.02 m tris base , 0.5 m nacl , ph 7.5 ) for 1 hour at room temperature .
the blots were washed three times with tbs containing 1% tween 20 and incubated overnight at 4c with biotinylated pna ( sigma - aldrich ) ( 10 g / ml ) and with the sera from patients with gbs ( 1:100 ) .
after washing , the strips were incubated with avidin conjugated with peroxidase ( 1:1000 ) , and with anti - human igg antibodies conjugated with peroxidase ( 1:500 ) for 1 hour at room temperature .
visualization of the reaction products was done using dab , 15 mm imidazole , and 0.025% h2o2 in water ( biorad laboratories , hercules , ca , usa ) .
isolated glycoproteins were tested on western blot for their reactivity with pna , as a marker for gal - galnac determinant and with gm1-positive sera from patients with gbs .
pna staining of the separated glycoproteins isolated from the human peripheral nerve and c. jejuni ( o:19 ) indicate the presence of the gal - galnac determinant in several proteins [ figure 1 ] .
western blot analysis of the reactivity of isolated proteins to sera from patients with gbs , revealed positive reactivity of one main protein band with mobility between 60 and 70 kda , present in both isolates , with tested sera from patients with gbs [ figure 2 ] .
figure 2 also shows positive reactivity of bands with molecular weight lower than 60 kda in both isolates .
further studies are needed to determine whether these bands are degradation products of the main cross - reactive proteins , or are independent proteins that show positive reactivity with sera from patients with gbs .
western blot of isolated glycoproteins from human peripheral nerve ( lane 1 ) and c. jejuni ( lane 2 ) stained with biotinylated pna .
western blot of isolated glycoproteins from human peripheral nerve ( 1 ) and c. jejuni ( 2 ) stained with gm1-positive serum from patient with gbs .
after electrophoretic separation of the glycoproteins isolated from the human peripheral nerve and from c. jejuni ( o:19 ) , a reference gel strip cut from the gel was stained with 0.1% amido black , 1% acetic acid , and 40% methanol in water [ figure 3 ] .
bands corresponding to the proteins with mobility of the cross - reactive glycoproteins ( between 60 and 70 kda ) , as compared to standard , were cut from the unstained gel and used for further enzymatic digestion .
sds - page of isolated glycoproteins stained with 0.1% amido black , 1% acetic acid , and 40% methanol in water .
lane 1 : human peripheral nerve ; lane 2 : c. jejuni ( o:19 ) ; lane 3 : protein standard .
the cross - reactive glycoproteins from human peripheral nerve and c. jejuni ( o:19 ) were enzymatically digested with trypsin and obtained peptides were separated on sds - page and visualized with silver stain [ figure 4 ] .
bsa served as a control for digestion and a well without protein served as blank .
sds - page of the digested glycoproteins from c. jejuni ( lane 2 ) and peripheral nerve ( lane 3 ) stained with silver stain .
lane 1 : bsa ; lane 4 : blank ; lane 5 : protein standard .
western blot analysis of the separated peptides obtained after trypsin digestion revealed positive reactivity of several bands to pna , present in both digests from the peripheral nerve and c. jejuni ( o:19 ) , indicating the presence of the gal - galnac residue in these bands [ figure 5 ] .
after incubation with serum from patient with gbs associated with c. jejuni ( o:19 ) infection , bands showing positive reactivity were revealed in both digests [ figure 5 ] .
serum from healthy individual did not show any reactivity to the obtained peptides ( not shown ) .
western blot of the digested glycoproteins from peripheral nerve ( lane 2 ) and c. jejuni ( o:19 ) ( lane 3 ) stained with : ( a ) gm1 positive serum from patient with gbs , and ( b ) biotinylated pna .
infection by c. jejuni pen 19 that bears a gm1-like lipopolysaccharide ( lps ) induces high production of igg1 and igg3 anti - gm1 antibodies .
anti - gm1 antibodies bind to the nodes of ranvier and motor nerve terminal axons , inhibit motoneuron excitability , and produce muscular weakness .
pathologic studies of early cases of aman have shown deposits of activated complement components and immunoglobulins at nodes of ranvier motor fibers .
the disruption of paranodal region allows the entry of complement and immunoglobulins along the axolemma , with subsequent recruitment of macrophages , which invade the periaxonal space , leading to wallerian degeneration of motor fibers .
the immunopathology of gbs associated with c. jejuni infection is caused by a mechanism of molecular mimicry between c. jejuni lps and gm1 ganglioside of the peripheral nerve . a single c. jejuni strain
is shown to have several lpss with various ganglioside epitopes , and different penner 's serotypes have the same ganglioside epitope .
these indicate that penner 's serotyping system is not dependent on ganglioside - like lpss . on the other hand ,
some anti - gm1 antibodies may be gm1-monospecific , whereas others may cross - react with other gangliosides or glycoproteins .
the subtypes of anti - gm1 antibodies cross - reactive with neural glycoproteins may correlate with different gbs variant .
our results showed positive cross - reactivity of the peptides from the human peripheral nerve and c. jejuni recognized by gm1-positive gbs serum associated with c. jejuni infection .
we have previously shown that gm1 antibodies cross - react with several gal - galnac bearing glycoproteins at the node of ranvier in the peripheral nerve .
two of these glycoproteins have been identified as oligodendrocyte myelin glycoprotein ( omgp ) localized at the paranodal region and hyaluronate - binding domain of versican localized at the nodal gap . a hyaluronic acid - like repeating unit of lps is proved to be an antigenic determinant of o : 19 .
the structural similarity exists between hyaluronic acid - like repeat unit of c. jejuni ( o:19 ) and hyaluronate - binding domain of versican .
this supports the hypothesis that pathology in gbs may be mediated by cross - reactive autoantibodies directed against the gal - galnac epitope of ganglioside gm1 and hyaluronate - binding domain of versican , whose production is triggered by homologous antigen in c. jejuni ( o:19 ) .
the results of this study showed the presence of cross - reactive determinants in glycoproteins from the human peripheral nerve and c. jejuni ( o:19 ) , recognized by pna , and by gm1-positive serum from patient with gbs , associated with c. jejuni infection .
these findings indicate the possible molecular mimicry between isolated cross - reactive glycoproteins and its role in the development of gbs in patients with previous infection with c. jejuni .
further studies are needed to characterize and determine the molecular structure of the obtained cross - reactive glycoproteins and their epitopes .
elucidation of the molecular structure and function of the cross - reactive glycoproteins present in the human peripheral nerve and c. jejuni will indicate their role in the pathogenesis of gbs and also help in understanding the mechanisms that trigger the immune responses in postinfectious autoimmune diseases .
these data indicate the possible molecular mimicry between the cross - reactive glycoproteins present in c. jejuni and human peripheral nerve and also its potential role in the development of gbs following infection with c. jejuni ( o:19 ) . | objective : antibodies to ganglioside gm1 are associated with guillain - barr syndrome ( gbs ) in patients with serologic evidence of a preceding infection with campylobacter jejuni . molecular mimicry between c. jejuni lipopolysaccharide ( lps ) and ganglioside gm1
has been proven to be the immunopathogenic mechanism of the disease in the axonal variant of gbs .
gm1-positive sera cross - react with several gal - galnac - bearing glycoproteins from the human peripheral nerve and c. jejuni ( o:19 ) .
this study aimed to examine the immunoreactivity of the digested cross - reactive glycoproteins isolated from the human peripheral nerve and c. jejuni ( o:19 ) with peanut agglutinin ( pna ) as a marker for the gal - galnac determinant , and with sera from patients with gbs.materials and methods : for this purpose , the cross - reactive glycoproteins from peripheral nerve and c. jejuni ( o:19 ) were enzymatically digested with trypsin and the obtained peptides were incubated with pna and gbs sera.results:western blot analysis of the separated peptides revealed several bands showing positive reactivity to pna and to sera from patients with gbs , present in both digests from peripheral nerve and c. jejuni ( o:19).conclusions : these data indicate the possible molecular mimicry between the cross - reactive glycoproteins present in c. jejuni and human peripheral nerve and its potential role in the development of gbs following infection with c. jejuni ( o:19 ) . |
integrins are a type of cell membrane receptor protein composed of various and subunits , among which the v subunit is highly expressed on the surface of hepatic cell carcinoma ( hcc ) .
the v subunit is critically important during the infiltration , metastasis , and neovascularization of tumor cells .
it is reported that targeting of alpha - v integrins reduces malignancy of bladder carcinoma .
currently , research on targeted therapies aiming at integrins include monoclonal antibodies against integrins , integrin antagonists , and targeted modulation of integrin genes , among which integrin antagonists are mainly express in the artificially synthesized polypeptides containing poly ( rgd ) structures , such as cilengitide [ 35 ] .
gene therapy studies have reported on the antisense technologies and rna interfering technologies of oligonucleotides [ 611 ] .
use of radionuclides in internal radiation therapies has been a research emphasis of tumor therapies , among which some radionuclides decayed through electron capture , like i , emitting many low - energetic auger electrons and coster - kroning ( ck ) electrons during its decay process .
however , there is little published data on i marked antisense oligodeoxynucleotide ( asodn ) and small interfering rna ( sirna ) .
sirna is easily cleared by the kidneys , it is degraded by enzymes , the cell uptake rate is low , and the markers on it are easy to drop , which limit its wide usage .
thus , i was marked on asodn in this study . as a pei derivative ,
polyethylenimine - arg - gly - asp ( pei - rgd ) is a receptor - dependent nonviral vector . after pei undergoes complexation with nucleic acids and its analogues and rgd binds to the v subunit , pei - rgd together with nucleic acids and
its analogues enter cells through pinocytosis . in this study , the v subunit of antisense oligodeoxynucleotide ( asodn ) was marked by radionuclide i. after complexation with pei - rgd , the pei - rgd / i-(v ) asodn compound was introduced into hepg2 cells via receptor - mediated transfected .
we analyzed inhibitory effects of the compound on the growth and invasion of hepg2 cells and discussed the integration of antisense technology , rgd inhibitors , and nuclide internal irradiation in hcc treatment .
the integrin v subunit asodn ( 5-gccctccttccacaatccca-3 ) sequence was examined using blast and was found to be nonhomologous with other human gene coding sequences .
purification through polyacrylamide gel electrophoresis ( page ) and the generation of phosphorothioates at the 5 and 3 ends were performed by shanghai sangon biotech co. , ltd ( shanghai , china ) .
pei - rgd was purchased from polyplus - transfection . the innocyte cell invasion assay kit ( qia129 )
approximately 20 g of ( v ) asodn was dissolved in 0.1 m ammonium acetate solution ( ph 5.0 ) and heated at 60c for 30 min .
the mixture was then added successively to 10 l of 0.25 mm ki , 10 l of 5 mci nai , and 100 l of 0.03 m tlcl3 , mixed evenly and heated at 60c for 45 min . after cooling at room temperature , 5 l of 0.1 m na2so3 and 20 l of 1 m ammonium acetate solution
were added successively ; then , the mixture was adjusted to ph 7.0 and heated at 60c for 60 min .
using acetone : acetic ether ( 1:1 , v / v ) as a developer , the marking ratio was computed using paper chromatography . using 0.01 m hydroxyethyl piperazine ethanesulphonic acid ( hepes , ph 7.4 ) as an eluent
the radiochemical purity of the purified reactants was separately measured at 24 , 48 , 72 , and 96 h , and the stability was observed in vitro .
the hcc strain hepg2 was kindly provided by the model organism open laboratory at the college of life science and technology , tongji university .
the strain was cultured in dulbecco s minimum essential medium ( dmem ) with 10% fetal bovine serum in an incubator with 95% humidity and 5% co2 at 37c .
cells were grown to logarithmic phase , and drugs were delivered when 5060% of the cells adhered to the walls of the culture flask .
the cells were transfected with pei - rgd ( l ) : i-(v ) asodn ( g , 2:1 ) .
log - phase hepg2 cells were adjusted with dmem culture solution to 100 000 cells / ml and then inoculated into 96-well plates , with each mesh containing 500 l .
we established 5 study groups ( 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g pei - rgd / i-[v ] asodn ) and used a i-(v ) asodn group and a i group as the controls .
the cells were cultured in a co2 incubator for 48 h , and when 5060% of the cells adhered to the bottom of the plates , the drugs were delivered .
the cell suspensions were collected into centrifuge tubes to measure the total counts per minute cpm(a0 ) .
the dose - effect tests included 5 pei - rgd / i-(v)asodn groups : 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g .
seven groups were used for the control tests : a pei - rgd / i-(v)asodn group ( 2 l/1 g ) , a i-(v)asodn group , a ( v)asodn group , a pei - rgd/(v)asodn group , a pei - rgd group , a i group , and a blank group .
after transfection ( in the same manner described above ) , the cell inhibition rate was computed using the methyl thiazolyl tetrazolium ( mtt ) method . using the cytotoxicity test as a comparison
, cell invasion was tested using the innocyte cell invasion assay kit ( qia129 ) .
approximately 300400 l of serum - free culture solution was added to the upper chamber .
after incubation for 3060 min , approximately 500 l of 10% foetal bovine serum medium was added to the lower chamber . then
, 300350 l of the transfected cell suspension was added to the upper chamber and placed into a co2 incubator for 48 h. five hundred microliters of cell staining solution was added to the lower chamber .
the cells that adhered to the bottom side of the upper chamber were knocked into the lower chamber and cultured for another 30 min .
approximately 200 l of the cells in the staining solution was used for fluorescence detection , photographing , and calculation of the inhibition rates .
all data were processed with spss , version 19.0 ( ibm corporation , armonk , ny , usa ) using analysis of variance ( anova ) and multi - sample paired - comparisons . the results are expressed as means standard deviation .
the integrin v subunit asodn ( 5-gccctccttccacaatccca-3 ) sequence was examined using blast and was found to be nonhomologous with other human gene coding sequences .
purification through polyacrylamide gel electrophoresis ( page ) and the generation of phosphorothioates at the 5 and 3 ends were performed by shanghai sangon biotech co. , ltd ( shanghai , china ) .
pei - rgd was purchased from polyplus - transfection . the innocyte cell invasion assay kit ( qia129 )
approximately 20 g of ( v ) asodn was dissolved in 0.1 m ammonium acetate solution ( ph 5.0 ) and heated at 60c for 30 min .
the mixture was then added successively to 10 l of 0.25 mm ki , 10 l of 5 mci nai , and 100 l of 0.03 m tlcl3 , mixed evenly and heated at 60c for 45 min . after cooling at room temperature , 5 l of 0.1 m na2so3 and 20 l of 1 m ammonium acetate solution were added successively ; then , the mixture was adjusted to ph 7.0 and heated at 60c for 60 min .
using acetone : acetic ether ( 1:1 , v / v ) as a developer , the marking ratio was computed using paper chromatography . using 0.01 m hydroxyethyl piperazine ethanesulphonic acid ( hepes , ph 7.4 ) as an eluent
the radiochemical purity of the purified reactants was separately measured at 24 , 48 , 72 , and 96 h , and the stability was observed in vitro .
the hcc strain hepg2 was kindly provided by the model organism open laboratory at the college of life science and technology , tongji university .
the strain was cultured in dulbecco s minimum essential medium ( dmem ) with 10% fetal bovine serum in an incubator with 95% humidity and 5% co2 at 37c .
cells were grown to logarithmic phase , and drugs were delivered when 5060% of the cells adhered to the walls of the culture flask .
the cells were transfected with pei - rgd ( l ) : i-(v ) asodn ( g , 2:1 ) .
log - phase hepg2 cells were adjusted with dmem culture solution to 100 000 cells / ml and then inoculated into 96-well plates , with each mesh containing 500 l .
we established 5 study groups ( 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g pei - rgd / i-[v ] asodn ) and used a i-(v ) asodn group and a i group as the controls .
the cells were cultured in a co2 incubator for 48 h , and when 5060% of the cells adhered to the bottom of the plates , the drugs were delivered .
the cell suspensions were collected into centrifuge tubes to measure the total counts per minute cpm(a0 ) .
the dose - effect tests included 5 pei - rgd / i-(v)asodn groups : 2/1 , 4/2 , 8/4 , 16/8 , and 32/16 l/g .
seven groups were used for the control tests : a pei - rgd / i-(v)asodn group ( 2 l/1 g ) , a i-(v)asodn group , a ( v)asodn group , a pei - rgd/(v)asodn group , a pei - rgd group , a i group , and a blank group .
after transfection ( in the same manner described above ) , the cell inhibition rate was computed using the methyl thiazolyl tetrazolium ( mtt ) method .
using the cytotoxicity test as a comparison , cell invasion was tested using the innocyte cell invasion assay kit ( qia129 ) .
approximately 300400 l of serum - free culture solution was added to the upper chamber .
after incubation for 3060 min , approximately 500 l of 10% foetal bovine serum medium was added to the lower chamber . then
, 300350 l of the transfected cell suspension was added to the upper chamber and placed into a co2 incubator for 48 h. five hundred microliters of cell staining solution was added to the lower chamber .
the cells that adhered to the bottom side of the upper chamber were knocked into the lower chamber and cultured for another 30 min .
approximately 200 l of the cells in the staining solution was used for fluorescence detection , photographing , and calculation of the inhibition rates .
all data were processed with spss , version 19.0 ( ibm corporation , armonk , ny , usa ) using analysis of variance ( anova ) and multi - sample paired - comparisons .
i-(v)asodn was purified and placed at 37c , and the radiochemical purity at 0 , 24 , 48 , 72 , and 96 h was 96.681.38% , 95.611.56% , 90.571.95% , 87.381.67% , and 77.791.86% , respectively .
the radiochemical purity after 48 h was still greater than 90% , but it obviously decreased after 96 h. thus , we suggest that further experiments with this marker should be finished within 48 h after marking . the cell intake rates of the pei - rgd / i-(v)asodn groups were 10.921.20% ( 2 l/1 g ) , 12.770.85% ( 4 l/2 g ) , 7.781.37% ( 8 l/4 g ) , 7.111.76% ( 16 l/8 g ) , and 1.421.03% ( 32 l/16 g ) , 2.191.35% ( i-[v]asodn ) and 0.310.28% ( i ) . for the dose - effect tests , the inhibition rates of the pei - rgd / i-(v)asodn doses were 9.633.15% ( 2 l/1 g ) , 38.837.64% ( 4 l/2 g ) , 60.297.65% ( 8 l/4 g ) , 69.406.47% ( 16 l/8 g ) , and 92.644.41% ( 32 l/16 g ) ( figure 1 ) , with a correlation coefficient of r=0.879 . for the control tests , the inhibition rates are shown in table 1 and figure 2 .
the invasion test fluorescence fields for all groups are shown in figure 3 , and the inhibition rates are listed in table 2 and figure 4 .
the inhibition rate in the pei - rgd / i-(v ) asodn group was significantly greater than those in the other groups ( p<0.01 ) , indicating the greater inhibitory effect of pei - rgd / i-(v ) asodn on hepg2 cell invasion .
i-(v)asodn was purified and placed at 37c , and the radiochemical purity at 0 , 24 , 48 , 72 , and 96 h was 96.681.38% , 95.611.56% , 90.571.95% , 87.381.67% , and 77.791.86% , respectively .
the radiochemical purity after 48 h was still greater than 90% , but it obviously decreased after 96 h. thus , we suggest that further experiments with this marker should be finished within 48 h after marking .
the cell intake rates of the pei - rgd / i-(v)asodn groups were 10.921.20% ( 2 l/1 g ) , 12.770.85% ( 4 l/2 g ) , 7.781.37% ( 8 l/4 g ) , 7.111.76% ( 16 l/8 g ) , and 1.421.03% ( 32 l/16 g ) , 2.191.35% ( i-[v]asodn ) and 0.310.28% ( i ) .
for the dose - effect tests , the inhibition rates of the pei - rgd / i-(v)asodn doses were 9.633.15% ( 2 l/1 g ) , 38.837.64% ( 4 l/2 g ) , 60.297.65% ( 8 l/4 g ) , 69.406.47% ( 16 l/8 g ) , and 92.644.41% ( 32 l/16 g ) ( figure 1 ) , with a correlation coefficient of r=0.879 .
for the control tests , the inhibition rates are shown in table 1 and figure 2 .
the invasion test fluorescence fields for all groups are shown in figure 3 , and the inhibition rates are listed in table 2 and figure 4
. the inhibition rate in the pei - rgd / i-(v ) asodn group was significantly greater than those in the other groups ( p<0.01 ) , indicating the greater inhibitory effect of pei - rgd / i-(v ) asodn on hepg2 cell invasion .
the integrin v subunit is highly expressed in several types of malignant tumour cells ( e.g. , hcc and melanoma ) [ 1416 ] , and it is involved in the recognition of ligands , mainly through the rgd in its homologous repeated sequence identification ligands .
the integrin v subunit interacts with several ingredients in the extracellular matrix ( ecm ) , such as vitronectin , fibronectin , and fibrinogen .
it is excited by extracellular growth hormone and/or ecm signals , and it alters the proliferation , differentiation , and migrating abilities of tumor cells by activating ras / map and other signal pathways .
the integrin v subunit promotes the growth of tumor cells , inhibits their apoptosis , and controls their invasion and metastasis .
reported a human monoclonal antibody 264rad targeting v6 integrin reduced tumor growth and metastasis , and modulated key biomarkers in vivo .
existing research concerning the treatment of hcc by the integrin v subunit focuses on immunotherapy and gene therapy ; these methods have partially entered the stage of clinical drug trials . as previously reported , lm609 and vitaxin , the monoclonal antibodies of integrin v3 , both inhibited the invasion of tumors in vivo . in gene therapy , tumor - specific asodn or nuclear enzymes
are primarily used to reduce or alter the expression configuration of the integrin v subunit and interfere with v - subunit - mediated adhesion , movement , and transfer . because most of the selected transfecting vectors are viruses or liposomes , problems will likely emerge , such as insecurity and poor specificity of target cells in vivo , as well as the use of time - consuming and complex steps ( e.g. , vector cloning , sequencing , and validation ) .
thus , these vectors have not been shown to be favorable for use in vivo experiments and clinical applications .
therefore , in the present study , we used receptor - mediated gene transfer technology ; namely , the asodn was introduced into cells through selective combination between the vector pei - rgd and hcc membrane surface .
1 ) pei is a cationic compound that can bind via ionic bonds with asodn ; it carries asodn and absorbs h in the lysosome , thus inactivating nucleic acid enzymes in the lysosome and protecting asodn from degradation .
2 ) the pei surface is occupied by many short rgd polypeptides , such as the integrin v subunit , that are natural ligands .
these polypeptides specifically bind with v and enter target cells through endocytosis , thus realizing the targeted transfection .
reported cyclic rgd - poly(ethylene glycol)-polyethylenimine was more suitable for glioblastoma targeting gene transfer in vivo . in the present study ,
the cell intake rate was maximized at 4 l/2 g pei - rgd / i-(v ) asodn , and it decreased with increased dosage , likely because the receptor v was saturated on the hepg2 cell surface or the compound was toxic to the cells . therefore ,
the results showed that , with the increased dosage , the compound inhibited hepg2 cells in a concentration - dependent manner .
the latter results also correlated with the studied dosage range ( r=0.879 ) . because of the high cytotoxicity under high dosage and to reduce the effects during paired comparison , we selected the smallest dosage of 2 l/1 g for the control group .
1 ) the inhibition rates were not significantly different between the pei - rgd / i-(v)asodn group and the pei - rgd/(v ) asodn group ( p>0.05 ) , but both groups were differed significantly from the other groups ( p<0.001 ) .
additionally , at a low dosage ( 2 l/1 g ) , the toxic effects of the pei - rgd / i-(v)asodn group and the pei - rgd/(v)asodn group were not significantly different , but they were higher than in the other groups , indicating that the radiation bioeffect of i was not obvious inside the cells .
2 ) the pei - rgd group , i-(v)asodn group , ( v)asodn group , and i group did not differ significantly from the control group ( p>0.05 ) , indicating that the 4 substances at low dosage were not cytotoxic .
the likely causes for this result include the following : low - dosage pei - rgd was not cytotoxic ; without the operation of a vector , the asodn transfection rate was low , leading to a low targeting capability ; and the marked i and free i could not enter the nucleus and , thus , could not demonstrate radiation bioeffects against dna . to reduce the effects of compound cytotoxicity on hepg2 cell invasion , we selected the lowest dosage ( 2 l/1 g ) for the cell invasion tests .
the results showed that the inhibition rate of the pei - rgd / i-(v)asodn group was significantly different from those of the other control groups , indicating that the inhibitory effect on hepg2 cell invasion was greater than those in the other groups .
1 ) the combination of pei - rgd with v on the surface of hepg2 cells reduced the probability of v binding with other natural ligands ( e.g. , other cells , ecm , and basal membranes ) , indicating the competitive inhibitory effect .
2 ) the combination of pei - rgd with v induced endocytosis into the cytoplasm , thereby degrading v and reducing the density of v on the hepg2 cell surface .
3 ) under the protection of pei - rgd , ( v)asodn entering cells avoided the degradation by nucleases and bound with v mrna , thereby interfering with the expression of v .
the marked i produced auger electrons and ck electrons via disintegration , forming local high - energy deposition within 10 nm ( 10-bp dna chains ) around the disintegration locus , which would induce non - repairable injuries , such as double - strand breaks ( dsbs ) . compared with the pei - rgd/(v)asodn group , the cytotoxicity of the pei - rgd / i-(v ) asodn group was not significantly different , but the inhibitory effect on hepg2 cell invasion was significantly different , which indicated that although the ionizing radiation of i did not significantly inhibit the growth of hepg2 cells , it weakened the invasion ability of the hepg2 cells .
moreover , the inhibition effects on cell invasion were significantly different between pei - rgd/(v)asodn and pei - rgd , but the differences were not very large , indicating that the antisense technology did not satisfactorily inhibit the invasion of the hepg2 cells .
therefore , rna interference technology should be used in future works , which is characterized by a higher inhibitory effect , longer working time , and greater stability .
this study showed that pei - rgd / i-(v)asodn can efficiently inhibit the growth and proliferation of hepg2 cells and can weaken their invasive ability .
although the radiation bioeffect of i was not obvious inside the cells , it weakened the invasion ability of the hepg2 cells .
we also found that the usefulness of antisense technology for inhibiting the invasion of hepg2 cells was limited . | backgroundto investigate the in vitro inhibitory effects of pei - rgd/125i-(v)asodn ( pei , polyethylenimine ; rgd , arg - gly - asp ; asodn , antisense oligodeoxynucleotide ) on the growth and invasion of hepg2 cells.material/methodsasodn of the integrin v - subunit was marked with 125i and underwent complexation with pei - rgd , a pei derivative .
next , pei - rgd/125i-(v ) asodn was introduced into hepg2 cells via receptor - mediated transfection , and its inhibition rate on hepg2 cell growth was tested using the methyl thiazolyl tetrazolium ( mtt ) method .
the effects of pei - rgd/125i-(v ) asodn on hepg2 cell invasion ability were evaluated using the boyden chamber assay.results1 ) the 125i marking rate of ( v ) asodn was 73.784.09% , and the radiochemical purity was 96.681.38% ( greater than 90% even after a 48-h incubation period at 37c ) , indicating high stability .
2 ) the cytotoxicity assays showed that the cell inhibition rates did not differ significantly between the pei - rgd/125i-(v)asodn group and the pei - rgd/(v ) asodn group , but they were both significantly higher than in the other groups and were positively correlated ( r=0.879 ) with the dosage within a certain range .
3 ) the invasion assays showed that the inhibition rate was significantly greater in the pei - rgd/125i-(v ) asodn group compared to the other groups.conclusionspei-rgd/125i-(v ) asodn can efficiently inhibit the growth and proliferation of hepg2 cells and can also weaken their invasive ability . |
an axillary mass was detected incidentally in a healthy 45-year - old woman who eight months earlier had undergone transabdominal hysterectomy and bilateral salpingo - oophorectomy due to uterine myoma .
mediolateral oblique imaging depicted a well - defined ovoid mass , 3 cm in size , in the left axilla ( fig .
gray - scale us using a broad band linear probe ( 5 - 12 mhz ) revealed a well - defined , uniformly hypoechoic , ovoid axillary mass , 323 cm in size and with good through transmission ( fig .
power doppler us of the mass showed prominent linear and branching peripheral blood flow ( fig .
precontrast , early phase , and delayed phase spiral ct scanning was performed prior to , 35 seconds after , and 180 seconds after , respectively , initiation of the intravenous infusion of 130 ml of nonionic contrast media at a rate of 2.0 ml / sec .
scanning parameters were 5-mm collimation , 9-mm table feed , and 7-mm reconstruction . at precontrast scanning , the mass was isodense to chest wall muscle ( fig .
1d ) , while postcontrast scans demonstrated rapid homogeneous enhancement and washout ( figs . 1e and 1f ) .
the mean density of the mass was 48 hu at precontrast scanning , 124 hu at the early phase , and 78 hu at the delayed phase .
ct depicted no lymph node enlargement in the mediastinum or abdomen . for us - guided biopsy ,
a 14-g automated gun was used : microscopic examination revealed tight concentric layering of lymphocytes at the periphery of the lymphoid follicles , with penetration by small capillaries , findings which were pathologically consistent with those of typical hyaline - vascular type castleman 's disease .
this showed that in the peripheral portion of the lymph node , the number and size of blood vessels were greater than in the central portion ( fig .
1 g ) , findings which correlated with those of power doppler us . in the germinal center ,
since castleman et al . ( 1 ) described 13 cases with the characteristic histopathologic features of a localized mediastinal lymph node hyperplasia in 1956 , many such cases have been reported under various names : lymphonodal hamartoma , follicular lymphoreticuloma , angiofollicular lymph node hyperplasia , angiomatous lymphoid hamartoma , and giant lymph node hyperplasia . the condition is currently classified into two major clinical subgroups : localized and disseminated castleman 's disease ( 3 ) , and two major histological types : hyaline - vascular and plasma - cell ( 4 ) .
keller et al . ( 4 ) found that 91% of all cases were the hyaline - vascular type and 9% were the plasma - cell type , while weisenburger et al .
( 5 ) reported that most cases of disseminated castleman 's disease were the plasma - cell type and in affected patients the prognosis was typically much worse than in those with localized disease .
although inflammatory and immunological processes are thought to be implicated , the exact etiology of localized castleman 's disease is unknown .
it is thought to represent either reactive lymphoid hyperplasia due to chronic antigenic stimulation by a virus , or a developmental growth disturbance ( 3 , 6 ) . in our case ,
the patient presented no symptoms and the axillary mass was detected incidentally . in castleman 's disease
, gray - scale us reveals a uniform hypoechoic mass ( 7 ) , while at doppler us , one that is hypervascular is depicted ( 8) . when differentiating between benign and malignant axillary nodes , shape and intranodal blood flow pattern are important . in a study by yang et al .
( 9 ) , gray - scale us showed that the mean longitudinal - transverse axis ratio ( sd ) was significantly lower in malignant ( 1.8 0.6 ) than in benign nodes ( 2.6 0.8 ) , while doppler us demonstrated that both the total number of vessels , and the number of those that were peripheral , were greater in malignant than in benign axillary nodes . in our case ,
the lymph node showed a low mean longitudinal - transverse axis ratio , 1.5 , and pathologic examination indicated that peripherally , many more and larger blood vessels were present , findings which suggested malignancy .
because mammography and us revealed no apparent abnormality , we believed that metastasis from another organ might have occurred .
lymphoid follicular dendritic cell dysplasia , which can become malignant , was diagnosed , and the observed peripheral hypervascularity may be related to this condition .
reactive lymph nodes tend to involve a diffuse histological process and are more likely to preserve a normal vascular pattern , with central hilar vessels ( 10 ) .
on the other hand , malignant changes caused by infiltrating tumor cells lead to the distortion and destruction of preexisting nodal vascular structures .
after central malignant infiltration of the lymph node , us - histopathologic correlation of the findings of lymphadenopathy demonstrated the presence of residual subcapsular vessels ( 10 ) . at ct
our patient underwent triphasic ct for evaluation of the hemodynamics of the lymph node , and postcontrast scanning demonstrated early rapid enhancement and washout . because rapid homogeneous enhancement of the node occurred ,
the imaging findings of castleman 's disease localized in the axilla were peripheral hypervascularity at power doppler us and early rapid enhancement at dynamic ct . | castleman 's disease is a rare benign lymphoproliferative disorder of uncertain origin which most commonly involves the mediastinum but rarely affects the axilla .
we report a case of localized castleman 's disease involving the axillary lymph node .
mammography revealed a well - defined , homogeneously dense ovoid mass , 3 cm in size , in the left axilla , while gray - scale ultrasonography ( us ) demonstrated a well - defined , uniformly hypoechoic ovoid mass with good through transmission .
peripheral hypervascularity was observed at power dopper us , and early rapid homogeneous enhancement at contrast - enhanced dynamic ct . |
autologous cell transplantation is a promising treatment option for large bone defects as it eliminates problems such as limited autologous bone availability , allogenic bone immunogenicity , and donor site morbidity and could be used for stabilizing loose alloplastic implant [ 1 , 2 ] .
marrow - derived stromal cells ( mscs ) alone and in combination with endothelial progenitor cells ( epcs ) have been shown to promote bone healing in a variety of different settings [ 35 ] .
the bone formation was shown to be improved when mscs were preconditioned with osteogenic substances such as ascorbic acid and dexamethasone .
another study demonstrated an enhanced callus formation after transplantation of osteogenic predifferentiated msc during distraction osteogenesis in 3 patients . in an animal study
dexamethasone - pretreated msc seeded on a collagen sponge result in a significant higher mineralization of the collagenous matrix .
this effect has been attributed to epc stimulation of early vascularization , a prerequisite for in vivo bone regeneration .
at least two major types of endothelial cell lines can be obtained by in vitro culture of mononuclear cells : first , the so - called endothelial - like cells or early epc and , second , the so - called outgrowth epc or late epc .
early epcs are derived presumably from monocytic / dendritic precursors , and some authors therefore designate them as endothelial - like differentiated pbmc [ 10 , 11 ] .
those cells can be generated in a sufficient amount within 3 to 5 days from a reasonable volume of blood .
early epcs are potent producers of vascular endothelial growth factor ( vegf ) . when transplanting msc and/or epc into bone defects , a scaffold is needed
. synthetic or processed bone - graft substitutes should be osteoinductive , enabling the osteogenic differentiation of cells , should provide appropriate mechanical stability , should permit the ingrowth of cells and vessels thereby improving bone regeneration , and should be resorbable .
hydroxyapatite ( ha ) sintered ceramics are widely used due to their osteoconductivity but their bioresorbability are comparatively low .
in contrast , tricalcium phosphate ( tcp ) ceramics were porous , resorbable , and biocompatible materials .
they do not provoke an inflammatory response and permit the ingrowth of cells and vessels during bone regeneration .
furthermore , tcp can be completely substituted for the bone tissue after stimulation of bone formation .
also the surface chemistry of the scaffold influences the behaviour , through either the influence of its charge density or atomic array on adherent or passing cell populations .
moreover , the surface charge of the biomaterials influences the binding of matrix proteins or growth factors , which might also influence the cell behaviour locally .
bone allografts consist of a collagen fibre network with attached hydroxyapatite crystals providing elasticity and perhaps osteoconductive properties to these scaffolds .
although antigenic structures were destroyed during processing components of the extracellular matrix , various growth factors such as bone morphogenetic protein-2 ( bmp-2 ) remain functionally active [ 21 , 22 ] .
bone graft substitutes are well described physically and chemically as an osteoconductive scaffold , but even more of interest are biological properties like cell adhesion and function of processed and synthetic biomaterials as delivery system for bone tissue engineering in critical size defects . however , depending on the method used to process the bone allografts the active ( osteoinductive ) and passive ( osteoconductive ) biologic interaction between the scaffold and the transplanted cells may vary and greatly influence the proliferation of the latter .
this was proved in a comparative study in which a markedly increased survival and metabolic activity of msc on human cancellous bone ( tutoplast ) in comparison to five different synthetic materials were observed .
recently it was shown that epc and msc can be successfully grafted onto a -tcp - matrix ; however , little data exists regarding the survival , proliferation , and differentiation of these cells when seeded on human cancellous bone ( tutoplast ) . a beneficial effect on bone healing mediated by osteogenic preconditioned msc has been repeatedly described [ 68 ] . but
moreover , the influence of osteogenic culture conditions on the number , metabolic activity , and differentiation of early epc alone or in coculture with msc has not been addressed yet . in the present study we investigated the influence of osteogenic culture conditions on the endothelial properties of human early epc
bone marrow cells were obtained from iliac crest aspirates of volunteer trauma patients ( n = 5 ) undergoing pelvic surgery .
this was performed in accordance with and with the approval of our hospital 's ethics committee .
briefly , mscs were isolated from fresh bone marrow aspirate using ficoll density gradient centrifugation ( 30 min , 1100 g , d = 1,077 g / ml , biochrom , berlin , germany ) . cells in the interphase were collected , washed twice with pbs containing 2% fetal bovine serum ( fbs ) ( 10 min , 900 g ) , resuspended in 3 ml mesencult + supplements ( cell - systems , st .
4 10 cells were seeded in a 25 cm culture flask and then expanded over three to five passages prior to being used in the present experiments .
cells were detached by 10 min incubation with accutase , then washed ( 10 min , 300 g ) , re - suspended in mesencult + supplements , and divided in 2 parts .
one part was adjusted to a density of 2.5 10 cells in 100 l and used for the present experiments , and the other was used for confirmation of msc , surface characteristics using flow cytometry ( facscalibur , bd - biosciences , heidelberg , germany ) .
mscs were negative for cd45 and cd34 and express cd73 , cd90 , and cd105 as described previously ; all antibodies were purchased from bd - biosciences .
briefly , pbmcs were isolated from buffy coat ( n = 5 ) by density gradient centrifugation ( 20 min , 600 g ) with ficoll ( 1.077 g / ml , biochrom , berlin , germany ) .
pbmcs were washed twice with cold pbs without ca and mg ( pbs , 10 min , 350 g ) , and 4 10 cells were cultivated on a fibronectin - coated ( 10 g / ml , sigma , deisenhofen , germany ) 24-well culture dish in 1 ml of endothelial basal medium ( ebm2 , cambrex , verviers , belgium ) supplemented with egm2 singlequots at 37c , 5% co2 . after 48 hrs , non- and weakly
adherent cells were removed , the medium was exchanged , and the cells were cultivated for an additional 72 hrs . a parallel preparation was performed to evaluate the percentage of endothelial cell - like differentiated cells .
cells were incubated for 1 h with 2.4 g / ml 1,1-dioctadecyl-3,3,3,3-tetramethylindo - carbocyanine - labeled acetylated low - density lipoprotein ( dildl , cellsystems , st .
cells were fixed with 2% paraformaldehyde for 10 min , and after washing with pbs fitc - labelled ulex europaeus agglutinin-1 [ 10 g / ml ] ( lectin , sigma , deisenhofen , germany ) was incubated for 1 h. cells presenting double - positive fluorescence were considered to be epc .
only preparations with a percentage of endothelial like differentiated cells of greater than 80% were used . for the determination of the expression of the endothelial markers cd31 and
von willebrandt factor ( vwf ) a volume of 100 l dildl prestained cells was incubated for 20 min with either monoclonal antibody against vwf ( dako , hamburg , germany ) followed by 15 min incubation with a fitc conjugated secondary antibody ( dako ) or with a fitc - conjugated monoclonal antibody against cd31 ( chemikon , hofheim , germany ) .
after two steps of washing , the cells were subjected to flow cytometry using a facscalibur ( bd biosciences ) .
cells were detached by incubation ( 10 min ) with accutase ( paa laboratories , linz , austria ) , washed once with mesencult + supplements ( cell - systems , st .
katharinen , germany ) and then adjusted to a density of 2.5 10 cells in 100 l . a processed human cancellous bone allograft material ( tutoplast , tutogen medical ) was used .
the solvent preserved , dried , and gamma - irradiated material has a particle size of 0.52 mm with a pore size ranging from 100 to 500 m .
the material has a high mechanical stability and a rapid biodegradability ( information provided by the manufacturer ) .
tutoplast granules were placed as a dense single layer in a 24-well plate ( nunc , wiesbaden , germany ) using sterile forceps and prewetted with 200 l pbs with ca and mg ( pbs ) . 5 10 mscs or epcs in a volume of 200 l were dripped onto the tutoplast layer and were incubated for 10 min at 37c .
when msc together with epcs were tested , 2.5 10 cells of each were mixed immediately prior to seeding .
after incubation , a medium containing the nonadhering cells was removed and dripped once again over the tutoplast layer , followed by incubation as indicated above .
the granules were then gently transferred to another well containing 500 l of a mixture consisting of 2 parts mesencult + supplements and 1 part ebm2+egm2 singlequots .
below this mixture is referred to as the medium and is described in .
the remaining cells in the supernatant and the bottom of the initial seeding well were isolated , counted , and the percentage of adhering cells was calculated ( ( initial cell number remaining cell number)/initial cell number ) 100 .
msc and epc , alone and combined , were seeded on tutoplast as described above .
approximately 4 - 5 granules were transferred to a single well of a 96-well plate using sterile forceps and incubated in 100 l medium for a period of 2 , 6 or 10 days in a co2 incubator at 37c .
parallel preparations included the same as above with the addition of substances that promote osteogenic differentiation : dexamethasone [ final concentration 1.0 10 m ] , -glycerolphosphate [ final concentration 1.0 10 m ] , and ascorbic acid [ final concentration 5 10 m ] .
all substances were obtained from sigma ( deisenhofen , germany ) . in order to detect msc and epc , msc 1 l of dildl ( cellsystem , st .
katharinen , germany ) or 1 l dapi [ final concentration 1 g / ml ] was added to the corresponding wells followed by further incubation for 1 h at 37c .
after three washes with pbs , the granules were transferred to a new well in order to prevent false - positive results caused by cells adhering to the bottom of the cultivation well .
finally , randomly chosen tutoplast granules were viewed at 100x magnification using fluorescence microscopy ( zeiss , axio observer , gottingen , germany ) , and the dildl - stained epc and dapi - stained msc were counted using cell explorer 2001 ( bioscietec , frankfurt , germany ) software .
tutoplast granules were seeded with either msc , epc , or both . after 2 days
the cell - tutoplast constructs were fixed in 2% glutaraldehyde in pbs ( merck , darmstadt , germany ) for 30 min and dehydrated by incubation for 15 min in pbs with increasing concentrations of ethanol ( 25% , 50% , 75% , 96% , and 100% ) .
subsequently the samples were incubated overnight in 1,1,1,3,3,3-hexamethyldisilazane ( merck , darmstadt , germany ) and slowly air - dried . the granules were fixed with carbon glue to an aluminium carrier and sputtered trice for each 1 min with gold ( agar sputter coater , agar scientific ltd .
a hitachi fe - sem s4500 was used ( hitachi , dsseldorf , germany ) .
the images were digitally recorded using the digital image processing system 2.6 ( point electronic , halle , germany ) . for determination of the metabolic activity as a correlate of the number of cells adhering to the tutoplast scaffold a cell proliferation kit i ( mtt , roche diagnostics , mannheim , germany )
, the mtt assay is based on the cleavage of the yellow tetrazolium salt mtt ( 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ) to purple formazan crystals by metabolically active cells .
the following combinations were performed : msc alone , epc alone , and msc and epc together .
cells were seeded on tutoplast , as described above , and 3 to 5 granules , depending on the granule 's size , were transferred to new wells in a 96-well plate ( nunc , roskilde , denmark ) and cultivated for 2 , 6 , and 10 days . before adding the mtt reagent , granules were transferred to an empty well , in order to prevent false positive results caused by cells adhering to the bottom of the well .
90 l of medium and 10 l of mtt labelling reagent were added to each well and cells were incubated for an additional 4 hrs .
the absorbance at 570 nm was then measured with an elisa reader ( ceres uv900c , bio - tek instruments , windoski , vt , usa ) . as controls ,
increasing numbers ( 1000 , 2500 , 5000 , and 10.000 ) of msc and epc were seeded directly in 96 well plates and assessed separately . in brief , total rna was isolated using the rneasy system ( qiagen , hilden , germany ) following the manufacturer 's instructions with the following exception .
approximately 50 l tutoplast - granules that had been sown with progenitor cells was incubated in rlt buffer for 3 min , the mixture was gently vortexed , and the supernatant was subjected to the rna isolation procedure .
the quality and quantity of rna was determined using the nanodrop nd-1000 device ( nanodrop technologies , wilmington , delaware , usa ) . contaminating genomic dna was removed by digestion with the rnase - free dnase kit following the manufacturer 's protocol ( qiagen ) .
each 250 ng of rna was reversely transcribed using an affinity script qpcr - cdna synthesis kit ( stratagene , la jolla , ca , usa ) following the manufacturer 's instructions .
real time rt - pcr was performed on a stratagene mx3005p qpcr system ( stratagene , la jolla , ca , usa ) .
pcr was performed using the primer assays for human collagen-1 ( col1a , nm_000088 , catalog number pph01299f ) , alkaline phosphatase ( alpl , nm_000478 , catalog number pahs-026 ) , core binding factor-1 ( cbfa-1 also known as runt - related transcription factor 2 , runx2 , nm_004348.3 , catalog number pph01897b ) , osteocalcin ( bglap , bone gamma - carboxyglutamate ( gla ) protein , nm_199173.3 , catalog number pph01898a ) , vascular endothelial growth factor ( vegf , nm_003376.4 , catalog number pph00251b ) , and von willebrand factor ( vwf , nm_000552.3 , catalog number pph02567e ) ; all purchased from biomol ( superarray , frederick , md , usa ) . as reference gene the expression of glyceraldehyde-3-phosphate dehydrogenase ( gapdh , nm_002046.3 , catalogue number pph00150e ) was measured .
relative quantification of the mrna levels of the target genes was determined using the comparative ct ( threshold cycle values ) method ( 2 method ) .
kruskal - wallis test with dunn 's post hoc test for multiplicity was used for comparisons between the groups and for the analysis of changes during the follow - up period ( day 2 versus day 6 and day 10 ) .
epc showed typical early epc characteristics such as spindle - shaped appearance , dildl uptake , binding of uea-1-lectin , and the expression of vwf and cd31 ( figures 1(a)1(d ) ) .
msc from all donors had a fusiform and a spindle - shaped appearance and demonstrated a typical pattern of surface markers ( cd34 , cd45 , cd71 + , cd90 + , cd105 + ) .
moreover , osteogenic differentiation was inducible as assessed by van kossa staining ( figures 1(d ) and 1(e ) ) .
the overall percentage of initially adhering cells ranged from 90% to 95% and did not differ significantly between msc and epc .
scanning electron microscopy images of tutoplast 2 days after cell seeding revealed a slightly roughened surface lacking sharp edges ( figure 2(a ) ) .
higher magnification revealed a surface composed of a dense layer of fibrils ( figure 2(b ) ) .
both msc and epc appeared firmly attached to the tutoplast surface . while msc had a bigger size than epc and showed a flattened and spread - out appearance ( figure 2(d ) ) , epc appeared elongated and spindle shaped ( figure 2(c ) ) . in the samples containing both msc and
epc their district shapes made it possible to differentiate between the 2 cell types on the tutoplast surface ( figure 2(e ) ) . in the samples containing both msc and epc , the number of epc declined significantly ( p < 0.05 ) from day 2 to day 6 and then remained stable until day 10 .
this was also observed in samples in which epcs were incubated in the presence of osteogenic supplements .
however , on day 2 after seeding , the number of adhering epcs was significantly higher in cultures supplemented with osteogenic factors ( figure 3(a ) ) .
the number of adhering mscs also decreased significantly ( p < 0.05 ) from day 2 to days 6 and 10 , independent of the presence of osteogenic factors ( figure 3(b ) ) .
the number of adhering cells paralleled the metabolic activity of epc , dropping significantly ( p < 0.05 , figure 4(a ) ) from day 2 to day 6 . in the presence of osteogenic supplements
the metabolic activity of epc slightly increased in comparison to controls ; however , this increase was not statistically significant ( figure 4(a ) ) .
the metabolic activity of msc decreased slightly with time , depicting a similar course as described for the number of adhering mscs .
in contrast to epc , when osteogenic substances were added to msc , metabolic activity was significantly increased compared to the corresponding controls on day 6 and day 10 ( figure 4(b ) ) .
the uptake of acetylated low - density lipoprotein is an active process that characterizes endothelial cells .
although a significant ( p < 0.05 ) decline of the dildl uptake was noted over the time , a significant ability of epc to uptake dildl was conserved over the entire 10-day observation period ( figure 5(a ) ) .
using an exposure time of 0.25 s the mean pixel brightness decreased from 60.0 5.5 at day 2 to 34 3.8 at day 10 ( p < 0.05 versus day 2 , figures 5(a ) and 5(c ) ) . under osteogenic conditions
the dildl uptake was impaired on day 2 ( trend : p < 0.07 ) and day 10 ( p < 0.05 ) in comparison to the corresponding controls ( figures 5(b ) and 5(c ) ) . in samples where msc and epc were cocultured
the mean pixel brightness between days 2 and 10 declined significantly ( p < 0.05 , figure 5(c ) ) .
however , the mean pixel brightness on day 2 was lower in comparison to epc cultured alone on tutoplast ( 37.1 2.2 versus 60.0 5.5 , p < 0.05 , figure 5(c ) ) . in co - culture and under osteogenic conditions
a comparable dildl fluorescence was observed , that declined significantly over 10 days ( p < 0.05 , figure 5(c ) , day 2 : 37.0 2.5 , day 6 : 30.5 2.6 , and day 10 : 18.5 2.0 ) . in order to assess msc and epc differentiation expression of osteogenic genes
( collagen-1alpha , runx-2 , bglap , alp ) and endothelial marker genes ( vwf , vegf ) were measured on day 10 . vwf gene expression significantly ( p < 0.05 ) increased when epcs were cultured on tutoplast versus the culture on plastic ( reference epc ) ( figure 6(a ) ) .
osteogenic stimulation of epc led to a decrease in vwf gene expression ( p = 0.09 , figure 6(a ) ) .
in contrast , vegf gene expression was significantly ( p < 0.05 ) higher in epc cultured on plastic as well as on tutoplast under osteogenic conditions versus epc cultured with tutoplast alone ( figure 6(b ) ) . collagen-1 and alp gene expression was limited to msc whereas a slight gene expression of runx and bglap was also observed in epc independent from the culture conditions .
collagen-1 gene expression on tutoplast was significantly ( p < 0.05 ) reduced in comparison to reference msc ( figure 6(c ) ) .
osteogenic conditions lead to a further significant reduction of collagen-1 gene expression of msc cultured on tutoplast ( p < 0.05 , figure 6(c ) ) .
in contrast , a trend towards increased collagen expression was observed in coculture with epc in comparison to msc cultured on tutoplast ( p < 0.09 ) .
furthermore , the collagen gene expression in the coculture was significantly elevated to msc , respectively , and msc and epc cultured under osteogenic conditions ( p < 0.05 , figure 6(c ) ) .
coculture under osteogenic conditions expressed higher collagen level in comparison to msc under osteogenic conditions .
the gene expression of alp ( alkaline phosphatase ) was significantly increased in reference msc in comparison msc cultured on tutoplast ( p < 0.05 , figure 6(d ) ) .
no differences regarding the gene expression of bglap ( osteocalcin ) and runx ( cbfa1 ) between the groups were observed ( figures 6(e ) and 6(f ) ) .
in the present study we demonstrated that epc can be cultured successfully on the processed human bone allograft tutoplast , alone and in combination with msc . a decline in the number of epcs and endothelial function ( dildl uptake ) was observed over the observation period but generally the endothelial properties measured were maintained , but to a reduced level .
the addition of osteogenic substances and the coculture with msc lead to an impairment of endothelial characteristics .
the number of adhering mscs also declined significantly over time whereas the metabolic activity increased significantly with osteogenic stimulation .
this was associated with a decrease of osteogenic differentiation ( reduced collagen-1 gene expression ) and an increase of vegf gene expression . in a recent study
the outcomes of treatment with processed cancellous bone allografts ( tutoplast ) versus autologous bone graft obtained from the iliac crest were compared in patients with comminuted distal radius fractures . over 70% of the patients had good to excellent outcomes in both groups .
this study demonstrated that processed human cancellous bone allografts can be as effective as autologous bone grafts for treating distal radius fractures . in another study cranioplasty using the tutoplast technology for autogenic bone processing was compared to conventional polymethylmethacrylate ( pmma ) calvarial reconstruction . in general comparable results were achieved with the autogenic bone but in young patients bone resorption occurred .
the favourable results with tutoplast in clinical settings attributed this effect to its natural surface characteristics . such a surface is thought to promote ingrowth , adherence , and migration of bone forming and other supplementary cells .
studies analyzing the influence of different biomaterials on cellular function demonstrate that cell - to - cell and cell - to - matrix interactions are key to bone formation [ 30 , 31 ] .
a recent in vitro study demonstrated that different methods of sterilizing bone grafts significantly alter msc adhesion , proliferation , and differentiation . in a former study and in the present study
we found that the number and metabolic activity of mscs adhering to tutoplast were superior to the synthetic materials tested .
our sem analysis revealed that tutoplast 's surface , consisting of a dense mesh of collagen fibrils ( figure 1(b ) ) , is suitable for the adherence of msc .
niemeyer and colleagues reported a higher seeding efficiency of msc on collagen-1 scaffolds coated with hydroxyapatite in comparison to a -tcp matrix .
they also observed that msc cultured on the collagen scaffold displayed a higher gene expression of bone sialoprotein , bmp-2 , alp , and osteocalcin when compared to msc cultured on uncoated -tcp .
we also found a significant improvement in adhesion and metabolic activity of msc cultured on a collagen scaffold ( tutoplast ) versus synthetic scaffolds such as -tcp and -tcp . in the present study we observed a significant decline in epc adherence to tutoplast over time .
the relative number of adhering cells ( as assessed by fluorescence microscopy ) was approximately halved over the 10-day observation period . during the same time
the observed decline of the dildl uptake was possibly due to the cultivation medium used . in order to meet the requirements of both epc and msc , that is , to maintain endothelial differentiation of epc and prevent endothelial differentiation of msc , the medium consisted of one part of egm + egm2 singlequots and two parts of mesencult .
the egm2 singlequots contains various growth and differentiating factors such as vegf and insulin - like growth factor ( igf ) . due to the addition of mesencult the concentrations of both factors
both factors were necessary to maintain the state of differentiation of epc and support the dildl uptake of epc ( vegf : , igf : ) .
in previous work we demonstrated that msc cultured with this mixture did not express endothelial characteristics with the exception of vegf gene expression .
interestingly , we observed a significant increase of vwf gene expression in epc on tutoplast , perhaps due to the collagen surrounding .
cultured early epc in collagen hydrogels for at least 14 days and observed a significant vwf expression .
however , no comparisons to other matrices were performed [ 25 , 35 , 36 ] . to our knowledge
no other report is available that describes the regulation of early epc cultivated on a dense 3d collagen-1 surface . in the present study we observed that the number of msc was equal under osteogenic conditions
this latter observation could be due to the transformation to an osteogenic phenotype which might be associated with a raised biosynthesis of peptides .
interestingly , the increase in metabolic activity was not accompanied by an elevated gene expression of collagen-1. it was reported for dermal fibroblasts that the collagen synthesis is negatively regulated by interaction with collagen fibers via a certain collagen receptor , and mesenchymal stem cells also express collagen receptors [ 37 , 38 ] .
thus , it is feasible to assume that the collagen biosynthesis underlies a similar negative feedback loop in msc seeded onto tutoplast .
osteogenic substances improve the bone forming capacity of msc but may harm the endothelial differentiation of epc .
the current study demonstrated that the endothelial differentiation of early epc on tutoplast is partly impaired under osteogenic conditions .
the dildl uptake was decreased under osteogenic conditions , and the gene expression of vwf tended to decline but remained still higher compared to reference epc cultivated on a fibronectin - coated surface .
the perpetuation of an endothelial cell - like phenotype under osteogenic conditions underlines the missing potential of early epc for osteogenic transdifferentiation . in one study the effect of osteogenic stimuli on cd133 expressing late epc
however as described in the introduction those cells differ in many respects from early epc used in the present work ( for review ) . in coculture
a significant decline of the vwf gene expression was observed in comparison to epc cultured alone on tutoplast . in coculture experiments a mixture consisting of each 2.5 10 msc and 2.5 10 epc was seeded ; in single culture experiments 5 10 epcs were used .
hence , the number of epcs was halved in comparison to single - culture experiments .
since the vwf - gene expression of msc is rather neglectable ( 10% of vwf gene expression in epc ) , it is feasible to assume that epcs are the main producers of vwf mrna in our coculture experiments .
since our own previous data demonstrated that early epcs do not express collagen-1 , the increase of collagen-1 mrna could be attributed to msc .
the increase of collagen-1 gene expression in our coculture experiments might be due to a release of osteogenic substances by epc .
they described the osteogenic stimulation of mscs which were cocultured with endothelial cells and identified bmp2 released by the endothelial cells as a responsible factor for the osteogenic differentiation . with regard to our study
it is feasible to assume that early epcs also release osteogenic substances ( i.e. , bmps ) or substances which foster the collagen synthesis ( i.e. , tgf- ) of msc .
however , if early epcs release bmp-2 and/or tgf- , this is still not known .
the possibility for cell - based therapy of large bone defects gains increased attention , and it has been demonstrated that the combination of msc and epc leads to a superior bone healing response in comparison to single - cell populations in different animal models [ 5 , 36 ] . additionally , other studies suggest an additional beneficial effect of an osteogenic differentiation of the msc prior the implantation to the host [ 68 ] .
however , less attention has been drawn to the carrier material to immobilize regenerative cells to the defect zone .
recent studies of our group suggest that processed cancellous bone chips are superior to synthetic bone grafts in terms of adhesion and function of msc .
the study provides evidence that the combined cultivation of two types of progenitor cells on a mineralized bone matrix for the purpose of tissue engineering is technically possible .
the early epcs maintain endothelial differentiation even under osteogenic conditions though endothelial activity was found to be reduced . moreover , our results suggest that concomitant early epcs stabilize the collagen-1 synthesis of msc which might be beneficial in bone healing
. however , wether msc and epc seeded together on a cancellous bone granules improve the bone healing has to be proved in experimental bone healing models . | introduction .
cancellous bone is frequently used for filling bone defects in a clinical setting .
it provides favourable conditions for regenerative cells such as msc and early epc . the combination of msc and epc results in superior bone healing in experimental bone healing models .
materials and methods .
we investigated the influence of osteogenic culture conditions on the endothelial properties of early epc and the osteogenic properties of msc when cocultured on cancellous bone .
additionally , cell adhesion , metabolic activity , and differentiation were assessed 2 , 6 , and 10 days after seeding . results .
the number of adhering epc and msc decreased over time ; however the cells remained metabolically active over the 10-day measurement period . in spite of a decline of lineage specific markers ,
cells maintained their differentiation to a reduced level .
osteogenic stimulation of epc caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression .
osteogenic stimulation of msc significantly increased their metabolic activity whereas collagen-1 and alkaline phosphatase gene expressions declined . when cocultured with epc ,
msc 's collagen-1 gene expression increased significantly .
conclusion .
epc and msc can be cocultured in vitro on cancellous bone under osteogenic conditions , and coculturing epc with msc stabilizes the latter 's collagen-1 gene expression . |
malaria parasites are capable of modulating the proportion of blood stage asexual parasites that develop into nonreplicating transmission stages ( gametocytes ) .
unfavorable conditions for asexual replication , and thus future transmission opportunities , stimulate an increase in short - term gametocytogenesis [ 13 ] .
many reports suggest that there is a trigger for the production of gametocytes and various factors have been proposed as possible promoting factors [ 4 , 5 ] .
carter and miller demonstrated that environmental conditions directly modulate the rate of gametocyte production by p. falciparum in culture .
we echo the authors suggesting that multiple factors are likely to promote gametocytogenesis : higher parasitemia , grade of synchrony of the culture , hematocrit of the culture , quality and age of the blood , addition of lysed erythrocytes , elimination of asexual stages by different methods ( sorbitol treatment or addition of pyrimethamine ) , media modification due to cell products and parasite metabolism , and so forth [ 4 , 711 ] .
these factors support the hypothesis that increased gametocyte production is a general response to retarded asexual growth in unfavorable and stressed conditions .
finally , it is crucial to keep a sufficient , yet fine balance , between inducing stressed conditions in asexual cultures ( to maximize gametocytogenesis ) and minimizing cell death [ 2 , 12 ] .
the aim of our study was to consider all these factors involved in gametocytogenesis to set up an optimized and efficient method whereby a reasonable number of young and mature p. falciparum gametocytes could be produced in vitro .
such a system is an absolute requirement for the development of in vitro assays measuring the gametocidal activity of new antimalarial drugs and would also constitute a starting point for the in vitro production of sporogonic stages .
rpmi 1640 with 25 mm hepes , sodium bicarbonate , and glutamine ( gibco ref : 52400 - 025 ) , supplemented with 10% of pooled human sera ab
( 14 - 490 e cambrex ) and 0.15 mm of hypoxanthine ( from ht supplement x50 , gibco ref .
human sera were decomplemented for 30 minutes at 56c , aliquoted and stored frozen at 20c until use .
red blood cell stock suspensions were prepared from whole - blood bags from incomplete blood donations , provided by the spanish red cross ( less than 25 days after sampling ) .
whole blood was centrifuged and washed 3 times with rpmi without serum by centrifugation ( 10 minutes at 2000 rpm ) .
washed red blood cells were kept as a 50% suspension in complete medium and stored for a maximum of 4 days at 4c .
p. falciparum 3d7 , dd2 , fcr3 , hb3 , w2 , and nf54 strains were obtained from the mr4 resource center ( atcc ) and maintained in complete medium at 5% of hematocrit value in continuous culture using a method adapted from trager and jensen .
gametocytemia was calculated by counting as a percentage the number of gametocytes ( determining each specific stage ) per number of erythrocytes ( 5000 rbcs ) .
thin blood films were made every day from each culture flask , fixed with methanol , and stained for 10 minutes in giemsa ( merck , ref : 1.09204 ) at 5% in buffered water ph 7.2 ( buffer tablets , merck ) .
the culture was maintained in 75 cm culture flasks ( canted neck , corning ) at 37c , in a low oxygen atmosphere ( 5% co2 , 5% o2 , 95% n2 ) .
to establish synchrony , cultures were treated by the method described by lambros and vanderberg based on sorbitol treatment .
5% ( w / v ) of sorbitol ( sigma s 6021 ) in cell culture grade water ( sigma w3500 ) was used .
prewarm an aliquot of 5% d - sorbitol , complete medium and rpmi at 37c .
remove most of the used medium from the flask and transfer the remaining culture to a 15 ml tube .
centrifuge for 5 minutes at 1800 rpm at room temperature and annotate the pellet volume .
transfer to a 50 ml tube and wash twice with prewarmed rpmi centrifuging for 10 minutes at 1800 rpm at room temperature .
after the last wash , remove as much supernatant as possible without aspirating the cells .
transfer pellet to a 15 ml centrifuge tube and wash with 10 ml of complete medium , remove supernatant , adjust pellet to 50% of hematocrit value , and use it to inoculate a culture in a 75 cm culture flask , adding fresh rbcs to maintain hematocrit value at 5% for synchronic cultures and at 2% for gametocyte production cultures . use the same procedure of synchronization described above , except the sorbitol pulse ( 2.5 volumes of pellet ) and the last step , in which the pellet is adjusted to 2% of hematocrit value without the addition of fresh rbc . remove vials from liquid nitrogen and thaw immediately in a 37c waterbath .
add 12% ( w / v ) nacl in pbs ( 0.2 ml per ml of stabilate ) , dropwise very slowly , constantly swirling tube .
add slowly 1.6% ( w / v ) nacl in pbs ( 10 ml per ml of stabilate ) .
add slowly 0.9% ( w / v ) nacl with 0.2% ( w / v ) glucose in pbs ( 10 ml per ml of stabilate ) .
remove supernatant and resuspend the pellet in 10 ml incomplete rpmi , adding it slowly at first .
resuspend cells in 6 ml complete rpmi culture medium , transfer to a 25 cm culture flask and incubate at 37c .
asexual parasite cultures were diluted to a 0.2% parasitemia and 6% hematocrit value with fresh rbc in a 75 cm culture flask ( day 0 ) . on day 3 , the cultures were diluted from a 6% to a 3% hematocrit with culture medium and then maintained for the next 18 days with daily medium changes .
giemsa - stained slides of the cultures were prepared daily to monitor parasitemia and gametocytemia .
asexual parasite cultures were diluted to a 1% parasitemia and 2% hematocrit value with hemoglobin - depleted rbcs in a 75 cm culture flask with 20 ml of culture medium .
[ hemoglobin depleted : red blood cells were incubated for 30 minutes with hypotonic solution ( 5 mm hepes , 11 mm glucose , 2 mm atp in bidistilled water ) to reduce their hemoglobin content ] .
giemsa - stained slides of the cultures were prepared daily to monitor parasitemia and gametocytemia .
cultures treated with two serial sorbitol treatments were subcultured at 0.5% or 1% of the initial parasitemia ( depending on the strain used ) and 2% of hematocrit value in a 75 cm culture flask with 20 ml of culture medium .
the culture medium was changed on days 4 and 6 and daily from days 8 to 15 . on days 9 , 10 , and 11 , 2.5 pellet volumes of 5% ( w / v ) sorbitol solution were added for 5 minutes once a day to remove asexual remaining forms and select only gametocytes .
giemsa - stained slides of the cultures were prepared daily to monitor parasitemia and gametocytemia .
combination of both protocols using the culture settings of chavalitshewinkoon - petmitr procedure complemented with the hemoglobin depleted rbcs from bennett protocol [ 7 , 11 ] .
asexual parasite cultures were synchronized and diluted to a 0.51% initial parasitemia and 10% hematocrit value in a 75 cm culture flask with 20 ml of culture medium .
after 48 hours of culture , lysed uninfected erythrocytes ( lue ) suspension was added at final concentration of 20% to induce gametocytogenesis .
giemsa - stained slides of the cultures were prepared daily to monitor parasitemia and gametocytemia . for lysis of erythrocytes ,
washed and pelleted human rbcs were frozen at 10c for 20 minutes and warmed up again at 37c for 20 minutes .
this was repeated twice . to reduce toxicity , we diluted one - part lysed erythrocytes with one - part rpmi medium .
asexual parasite cultures were synchronized and diluted to a 0.75% initial parasitemia and 2% hematocrit value in a 75 cm culture flask with 20 ml of culture medium .
, 40 l of pyrimethamine ( 24.87 ng / ml ) was added to eliminate asexual stages . on day 7 , the culture was washed twice with 40 ml of rpmi ( 1800 rpm , 5 minutes ) in order to reduce the drug amount .
giemsa - stained slides of the cultures were prepared daily to monitor parasitemia and gametocytemia .
combination of both protocols using the culture settings of chutmongkonkul procedure complemented with the addition of lue suspension at 20% on day 4 , from schneweis protocol [ 4 , 8 ] .
combination of both protocols using the culture settings of chavalitshewinkoon - petmitr procedure complemented with the addition of lue suspension at 20% on day 4 , from schneweis protocol [ 4 , 7 ] .
manipulation of the culture conditions and addition of pharmacological agents could increase gametocyte numbers or apparently initiate gametocytogenesis .
for example , the addition of red cell lysate [ 4 , 6 ] , presence of human serum , or some antimalarial drugs such as chloroquine could increase gametocyte production .
proposed that synchronized sexual development was induced by a sudden increase to the hematocrit of a fast growing ring stage culture .
affirmed that it is necessary for the asexual blood stage to reach high densities in the culture and become stressed to convert to the production of large numbers of gametocytes .
ifediba and vanderberg concluded that gametocyte production could be enhanced by reducing the hematocrit for part of the culture period .
buchholz et al . affirmed that sexual conversion could be induced in vitro by a sudden decrease in hematocrit concentration and a high parasite load , reflecting conditions of physiological stress in the human host that are known to correlate with increased numbers of transmission stages in blood circulation .
it is possible that there may not be a single mechanism and that multiple factors may contribute to the decision to form gametocytes .
considering some of these aspects , existing static culturing protocols that stimulate gametocytogenesis typically involve seeding flasks with a low parasitemia ( 0.2% ) and high hematocrit value ( 6% ) on day 0 of gametocyte production . on day 3
, the cultures were diluted from a 6% to 3% hematocrit ( increasing the medium volume to stimulate healthy growth of the induced gametocytes ) and the medium in the flasks was then changed daily over the next 1820 days without any addition of fresh uninfected erythrocytes , as gametocyte production is facilitated under these conditions [ 9 , 10 , 12 ] . moreover , schneweis et al . and bennett et al . proposed , respectively , that adding lysed erythrocytes ( at a final concentration of 20% ) to the culture medium or reducing the hemoglobin content of the erythrocytes using a treatment with hypotonic solution for 30 minutes would enhance in vitro gametocyte production [ 4 , 11 ] .
also suggests that not all laboratory strains produce gametocytes with the same occurrence even if these strains are stimulated by the same induction method .
another factor to consider is the correlation between asexual parasitemia and commitment to sexual development .
carter and miller concluded that environmental conditions directly modulate the rate of gametocyte production by p. falciparum in culture .
however , the actual factors responsible and the way in which they can modify the level of sexual conversion are still to be determined [ 2 , 3 ] . it is commonly accepted that , when the level of asexual parasitemia decreases , the rate of parasite conversion to gametocytes increases [ 2 , 5 ] . related to this , chutmongkonkul et al . described the use of pyrimethamine at low concentrations , 40 l of pyrimethamine ( 24.87 ng / ml ) , to eliminate asexual stages and chavalitshewinkoon - petmitr et al . proposed treatment with sorbitol ( at 5% w / v on days 9 , 10 , and 11 of gametocyte production ) for the inhibition and elimination of asexual parasites [ 7 , 8 ] .
considering all the factors that could modulate gametocytogenesis , we have compared and combined some of the protocols mentioned previously using different p. falciparum strains to establish the best in vitro approach for an efficient production of p. falciparum gametocytes from stages i to v. various conditions used in each experiment and their results are summarized in table 1 .
first , we compared the reduction of hematocrit , the influence of hemoglobin content and the decrease of asexual parasites by sorbitol treatment as enhancers of gametocytogenesis , using synchronous cultures of plasmodium falciparum strains 3d7 and dd2 ( experiment 1 ) [ 7 , 9 , 11 ] .
the results we obtained showed that reducing the number of asexual parasites in culture was the most efficient strategy to produce higher numbers of pure gametocytes .
in contrast , reduction of hematocrit did not produce the gametocytemia that had been described by previous authors [ 9 , 10 , 17 ] .
the influence of hemoglobin content was associated with hypergametocytemia within 2 to 4 days of gametocyte production .
although we cultured parasites with more hemoglobin depletion than bennett ( 40% of hemoglobin ) , we were unable to reach hypergametocytemia in either the 3d7 or dd2 strain and could not completely eliminate asexual parasites from the culture .
alternatively , we combined the decrease of asexual parasites with the reduction of hemoglobin content and analyzed the outcome of both approaches when applied alone or in combination .
the initial parasitemia of the gametocyte production culture was also investigated ( experiment 2 ) .
although the best results , considering the percentage of parasitemia ( 1.64% 0.24 ) , were obtained using the combined strategy , this approach was discarded as it produced a mix of asexual parasites and gametocytes .
in contrast , with the chavalitshewinkoon - petmitr protocol only gametocytes were observed , due to the elimination of asexual parasites by sorbitol treatment [ 7 , 11 ] .
the initial parasitemia of the gametocyte production culture must be established taking into account the synchrony and growth ratio of each strain in order to keep the balance between inducing stressed conditions and minimizing cell death . in previous studies ( data not shown )
, we realized that synchronic cultures with the highest initial parasitemia could compromise the reinvasion process and survival of the parasites .
suggest , low initial parasitemia increased the numbers of gametocytes meanwhile higher parasitemia produced fewer gametocytes .
considering the grade of synchrony and the growth ratio of the 3d7 and dd2 strains , we concluded that the best initial parasitemia on gametocyte production was 0.5% for these specific strains .
the production of gametocytes from different synchronized strains ( 3d7 , dd2 , fcr3 , hb3 , w2 , and nf54 ) using the chavalitshewinkoon - petmitr method ( with the modification of the initial parasitemia ) was also studied ( experiment 3 ) .
the highest gametocytemia was obtained with the nf54 strain ( 0.75% 0.20 ) . as suggested by bennett et al .
several authors affirm that recently isolated strains are more likely to form gametocytes than strains that have been in culture for long periods of time [ 21 , 22 ] .
mons and van der kaay affirmed that gametocyte generation of p. berghei increases after the cryopreservation of the cultures .
concluded that the potential for producing nf54 gametocytes is undiminished during storage of stabilates in the cryopreserved state . to test this statement
, we launched a new production of gametocytes according to the chavalitshewinkoon - petmitr protocol and using nf54 and w2 strains just released from cryopreservation ( experiment 4 ) .
as shown in table 1 , gametocyte numbers were higher when prepared from just thawed nf54 parasites .
we observed that nf54 is a strain that retains the capacity to produce gametocytes after being cryopreserved .
although there were no differences between synchronic and just thawed strains ( p value 0.06 for w2 and 0.37 for nf54 ) , we preferred to use storage stabilates of nf54 to avoid the reduction of gametocyte production due to passage numbers .
taking into account all the previous results , the reduction of asexual parasites was considered a key factor in promoting gametocytogenesis . to study this factor , we compared two protocols based on treatment by either sorbitol ( chavalitshewinkoon - petmitr protocol ) or pyrimethamine ( chutmongkonkul protocol ) for the reduction of asexual parasites of the culture ( experiment 5 ) [ 5 , 6 ] .
the original protocols of chavalitshewinkoon - petmitr and chutmongkonkul were run in parallel using the nf54 strain [ 7 , 8 ] and combining both treatments with the addition of lysed uninfected erythrocytes ( amplifying the accumulation of parasite metabolism and cell products ) as another factor that could influence the sexual development of the parasite ( schneweis protocol ) .
as described in table 1 , the highest gametocytemia was obtained with the new combination of protocols ( experiment 5 ) , which considered the decrease of asexual stages by sorbitol treatment and the addition of lysed uninfected erythrocytes as the main factors on gametocyte production .
on the other hand , the chutmongkonkul protocol ( which reduced asexual parasites through the addition of pyrimethamine ) had less effect ( on gametocyte production ) , even when it was supplemented by the addition of lue . with the new combined method
, we reached a gametocytemia of 1.02% 0.05 where asexual parasites were completely eliminated .
this successful approach was reproduced several times to assess its reliability using the nf54 strain where conditions of initial parasitemia ( 0.75% ) and hematocrit ( 2% ) were also adjusted ( experiment 6 ) .
we observed significant differences ( p value 0.0034 ) between this new method and basal production of gametocytes on a control experiment ( gametocyte production with no addition of lue or pulse sorbitol with the nf54 strain ) .
this optimized protocol combined two key factors shown to enhance gametocytogenesis : the addition of lysed uninfected erythrocytes ( lue ) and the complete elimination of asexual stages through sorbitol treatment .
we decided not to use the elimination of asexual stages through pyrimethamine treatment for two reasons : ( i ) it does not completely eliminate the asexual stages and ( ii ) as chutmongkonkul et al
. suggested , we observed that not all laboratory strains produce gametocytes to a similar extent even if they are stimulated by the same induction method .
the level of culture synchrony was increased by thawing the parasites and was an additional factor we integrated to improve the production of a healthy population of gametocytes with maturation capability , avoiding the reduction of gametocyte production due to passage number of the cultures . in summary , we compared previously described protocols and set up an optimized method for an improved in vitro production of p. falciparum gametocytes , which consisted of two main steps : thawing an aliquot of cryopreserved p. falciparum nf54 parasites ( that retain their capability of producing gametocytes ) and synchronizing the resulting culture by two serial sorbitol treatments ( 48 hours each ) in order to start gametocyte production with the highest amount of ring stages on the culture .
a certain percentage of the ring forms that appear in culture are committed to developing into gametocytes ; this percentage ( conversion rate ) was calculated using the following formula : [ number of stage ii gametocytes that appear in the culture 2 days after the ring forms ( per 5000 rbcs)/number of ring forms ( per 5000 rbcs)]*100 ; the conversion rate from rings to gametocytes was between 11% and 23% . at the end of the second sorbitol treatment , the culture
was adjusted to 0.75% of the initial parasitemia and a 2% hematocrit value ( day 1 of production ) . on day 4
, a suspension of lysed uninfected erythrocytes ( lue ) was added at a final concentration of 20% to induce gametocytogenesis .
the culture medium was changed on days 4 and 6 and daily from days 8 to 15 to remove cell debris .
sorbitol treatments ( 2.5 pellet volumes of sorbitol solution ) on days 9 , 10 , and 11 were carried out to remove remaining asexual forms and to enrich the cultures in gametocytes .
p. falciparum nf54 was clearly identified as the strain that produced the highest gametocytemia ( 1.18 0.07 ) with this protocol , producing the complete development of each gametocyte stage ( i v ) and excellent reproducibility .
the time course of p. falciparum nf54 gametocytes produced in vitro following the protocol described above is shown in figure 1 .
total parasitemia increased by 7 - 8% up to day 4 when , due to stress conditions , gametocyte production started to raise .
we observed that as asexual stages decreased on day 8 , the percentage of gametocytes increased until day 14 , reaching a gametocytemia of 11.2% . in vitro gametocyte production required approximately 15 days . during this time , it was possible to define the specific day each gametocyte stage appeared and their maturation from early ( young gametocyte ) to late ( mature gametocyte ) stages . on day 6 and day 11 of production , we obtained stage ii and stage iii , respectively .
the gametocytes then matured up to stage iv on day 14 and stage v on day 16 ( figure 2 ) .
we classified the gametocyte stages ( i v ) according to the description made by carter and miller .
considering key factors involved in gametocytogenesis , such as the reduction of asexual stages and the addition of lysed uninfected erythrocytes , here we propose an optimized and efficient strategy for the in vitro production of p. falciparum gametocytes based on the comparison of different protocols .
this new method will allow the accurate determination of the gametocidal activity of new antimalarial drugs against each developmental phase of the sexual blood stage ( study in progress , data not shown ) and will also be the starting point for the production of the subsequent sporogonic stages . | the generation of sexually committed parasites ( gametocytogenesis ) is poorly understood in malaria . if the mechanisms regulating this process were elucidated , new opportunities for blocking malaria transmission could be revealed .
here we compare several methods described previously for the in vitro production of plasmodium falciparum gametocytes .
our approach relies on the combination of several factors that we demonstrated as impacting on or being critical to gametocytogenesis .
an improved method has been developed for the in vitro production of p. falciparum gametocytes as the first step toward obtaining adequate numbers of pure gametocytes for in vitro studies , such as , for example , the identification of transmission blocking drugs . |
the account of the way canadian psychiatric mental health nursing ( pmhn ) has emerged into its current state can provide an insightful perspective that fosters a better understanding of its present challenges and opportunities .
this review paper examines the development of canadian pmhn in ontario and the western provinces since the beginning of the 20th century .
, canada had the highest growth rate of the g8 countries in the 2011/2012 period . in canada , registered nurses ( rns ) , registered psychiatric nurses ( rpns ) , licensed practical nurses ( lpns ) , and nurse practitioners are employed in the area of psychiatric or mental health care .
rpns are a regulated separate profession in the provinces of manitoba , saskatchewan , alberta , and british columbia ( for the purposes of this paper rpn refers to registered psychiatric nurses that are only legislated in the four western provinces . in ontario , registered practical nurses , also abbreviated to rpn , provide care in all settings and are not solely specific to or trained in the area of psychiatric mental health .
when rpn is used in this paper , the reference is to registered psychiatric nurses . )
statistics on the various types of nursing professions employed in psychiatric or mental health care can reflect pertinent conditions in relation to the canadian mental health system .
for instance , in 2010 , 5.1% of all rns in canada were working in the mental health care sector , a slight decrease from 2007 where 5.2 were employed in this same area . in ontario alone , between 1993 and 2003 , there has been a 29.4% drop in the number of nurses working in the psychiatric sector .
the number of rpns increased by 3.8% since 2007 but the rpn workforce remained at 1.5% .
the proportion of full time rpns in psychiatric nursing decreased from 67.8% in 2007 to 62.2% in 2011 .
the rpn profession also has the highest percentage of males , 22% , of all the canadian nursing professions .
according to the canadian nurses association , nurse practitioners ( nps ) account for 0.9% of the entire number of rns . of the 2,486 nps employed in canada ,
the statistics concerning the employment of nurses in the mental health care sector are meaningful when considered in light of the statistics concerning how many people in canada suffer from mental health problems .
the canadian community health survey by statistics canada was completed in 2003 and is scheduled again in 2013 .
this will enable further comparisons of how canada fairs in terms of prevalence of mental health disorders and the mental health services available .
in 2003 , it revealed that two - thirds of those who experience mental health problems do not receive mental health services .
furthermore , persons living with mental illness may have difficulty competing for limited health care due to high rates of poverty and disability . in 2012 , the institute for clinical evaluative sciences and public health ontario released the report opening eyes , opening minds : the ontario burden of mental illness and addictions report .
this report measures the extent of mental illnesses in ontario in comparison with the prevalence of other medical conditions .
addictions and mental illnesses represent an enlarging burden . the need for improved accessibility of mental health services
the size of the pmhn workforce and the accessibility of services may impact mental health care for canadians . historically , the deinstitutionalization trend that began in the 1960s resulted in many persons with mental illness being released into the community where a serious lack of mental health services prevailed .
this led to a significant portion of individuals with mental illness to become imprisoned in jails or detention centers with limited access to mental health care .
additionally , a revolving door phenomenon occurred where previously institutionalized individuals were readmitted into acute psychiatric care settings . to further understand the canadian context for pmhn , it is useful to be familiar with developments concerning mental health care in canada .
there are recent major initiatives underway in canada with regards to mental health reform . in 2005 ,
a national review of the canadian mental health system took place as outlined in the kirby report . in 2006 , the standing senate committee on social affairs , science and technology completed the final report entitled out of the shadows at last transforming mental health , mental illness and addiction services in canada .
as the mental health care system was considered to be fragmented , the final report made recommendations for the reformation of mental health care .
the assembly of a mental health commission to enable a national strategy for mental health care was emphasized by michael kirby , the chairman of the standing senate committee on social affairs .
funding for the commission came in 2007 , and in 2009 , the mental health commission of canada released the framework towards recovery and well - being : a framework for a mental health strategy for canada .
seven goals are set in this framework that represent how a transformed system will appear .
the goals depict that everyone should have the opportunity for optimum mental health and well - being .
the mental health commission has recognized that there is a lack of opportunities for canadians with mental health disorders to achieve optimum mental health .
this calls for key stakeholders , that is , groups interested in responding to people with mental health concerns , to address ways to further the provision of mental health care .
nurses are the largest group of healthcare providers , and nurses who provide mental health care are important stakeholders in meeting the mental health care needs of canadians . in 2012 ,
the mental health commission of canada released the blueprint changing directions , changing lives : the mental health strategy for canada .
six strategic directions are given in this report including the provision of mental health services and treatments accessible to the people who need them .
reference to the vital role nurses have in conjunction with the team of health care providers to fully achieve the strategic directions is not explicit in the 2012 report .
in addition , nps are becoming increasingly more common in providing primary health care within canada .
their role in the assessment , diagnosis , and treatment of mental health disorders may be extremely useful to further the strategic direction concerning enhanced accessibility to treatment .
collectively , nurses and nurse practitioners have a critical role in the strategic direction as identified by the mental health commission of canada .
a national strategy is needed , and mental health care providers will need to be included in this strategy according to the kirby report .
nurses who form the largest group of health care workers in canada may need to be strong stakeholders in the strategic direction for mental health care in canada .
the commission has yet to clearly voice how health care and nurses will be reorganized to support accessible services and treatments .
kirby indicates the pivotal position that mental health care providers have and also addresses concerns regarding the stress levels and mental health of health care professionals themselves .
indeed this leads to concerns regarding the quality of nursing work life for pmhn and how this specialty is responding to the demands of the mental health care system .
the analysis of the history of canadian pmhn can enable a better understanding of why pmhn is the way it is today .
with this understanding , opportunities for further pmhn development , education , and future research may surface . given the need for increased accessibility to mental health care and treatment , it is helpful to further explore how nursing will move to foster improved mental health care . at this time , there is a shortage of research studies that look at how the current nursing workforce specifically addresses improved accessibility to mental health care .
career pathways to become nps that specifically work in mental health are lacking , where nps may diagnosis and treat mental health conditions .
as an np working in mental health care , the options for pursing advanced mental health nursing education in canada are limited .
pmhn education varies across canada and unlike the united states there is no legislated psychiatric mental health nurse practitioner ( pmhnp ) role .
also rpns may wish to become advanced practice nurses or pmhnps that are able to diagnose or prescribe psychotropic medications .
a regulated pmhmp 's role may further the accessibility of mental health care in canada .
there are opportunities for nurses to achieve a master 's degree in mental health in manitoba ; however , there are no programs in canada that specifically yield pmhnps that may diagnose or prescribe medications particular to mental health care
. the educational programs for nurses also vary in each province in terms of content and the prerequisites . with the variety of different educational approaches to pmhn in canada , the career path to advance nursing practice in pmhn is complicated .
an rpn in the western provinces has had different preparation than an rn working in mental health care in ontario . how these diverse pmhn educational approaches have come to be and their implications for canadian pmhn can provide useful insights to inform future directions to pmhn in canada .
the purpose of this inquiry is to understand the current state of mental health nursing in canada through the intersections of history , gender , nursing education , and quality of work life as evidenced by the existing research and literature .
the research question that guided it was as follows : what does the research reveal about contemporary canadian pmhn in terms of the junctures of history , gender , education , and quality of pmhn work life ?
an integrative methodology was determined to be most suitable for this review due to the scarcity of data on the organization and efficacy of canadian pmhn .
the integrative review includes the stages of problem identification , literature search , data evaluation , data analysis , and presentation .
whittemore and knafl explain that the integrative review is particularly suited to inquiries with limited existing empirical research .
historical events in pmhn are relevant to how this field has come to be . yet
few research studies exist on canadian pmhn , and the studies are in the form of social historical analyses that reflect on the events of the past .
moreover , there is a paucity of data pertaining to canadian nps specific to mental health care and np mental health education .
sociological , critical , and social historical analyses , reports , and surveys on canadian nursing and canadian mental health care offered insight into the field of canadian pmhn that furthered this inquiry ( see tables 1 , 2 , and 3 ) . in the integrative review , according to whittemore and knafl , diverse methodologies may be incorporated to further a comprehensive grasp of issues suited to the complexities of health care .
the culmination , analysis , and evaluation of diverse data and research regarding pmhn enabled a perspective that may begin to further understanding mental health care in canada in light of the lacking research solely pertaining to canadian pmhn or pmhnps .
a sociological perspective allows for a critical assessment of common assumptions and fosters recognition of opportunities and constraints that shape our circumstances , the interplay between societal forces and personal lives , and of human diversity .
in addition , the concept of gender stratification is comprehensively analyzed within sociology and is applicable to the topic of this paper .
a sociological perspective is appropriate for this inquiry as nurses are employed within gendered hierarchal structures that interact and are influenced by the larger political context .
feminist theories , emerging from a sociological perspective , address patriarchy , power structures , and gender inequality .
they are concerned with the societal organization and interactions that maintain male authority and female subordination .
a feminist perspective enables the depiction of the social structures leading to the devaluation of women . social order then becomes the problem rather than women themselves .
the intent in this work is not to devalue women or nurses but , rather , to illuminate the social structures that have persevered throughout history and have influenced pmhn education and quality of work life .
wright calls this way of thinking the sociological imagination that grasps the connection between history and the way we are and the way we are becoming .
the history of pmhn seen through a sociological perspective can promote an understanding of its current state .
the databases included cinahl , psycinfo , evidence - based mental health , cochrane , pubmed , and proquest . published online journals were accessed through blackwell synergy and sage .
primary search results were then analyzed to search their references for secondary sources of interest .
the databases were accessed through the york university library , toronto , ontario and queens university library , kingston , ontario .
texts were borrowed through the assistance of the librarian at waypoint centre for mental health care , penetanguishene , ontario . through combinations of keywords
, topics searched for included psychiatric nurses in canada , canadian psychiatric nursing history , canadian psychiatric nursing education , canadian mental health nursing , canadian psychiatric mental health nursing , canadian nurses and gender , quality of life for canadian psychiatric mental health nurses , stress experienced by psychiatric nurses in canada , registered psychiatric nurses in canada , work stress of nurses working in mental health , literature review on psychiatric mental health nursing in canada , sociological perspective of psychiatric nursing , psychiatric mental health nurse practitioner , mental health nurse practitioner education , and sociology of nursing stress .
the searches included english language reports , studies , reviews , editorials , and narratives and was limited to canadian sources and canadian pmhn .
international comparisons were beyond the scope of this undertaking . with regards to the unique canadian historical influences shaping pmhn ,
there was a lack of studies that focused solely on the canadian pmhn experience as many reports included rpns and rns .
rns in the provinces east of manitoba do not have a separate psychiatric regulation , as do the western provinces .
no studies were found on the education in canada for nps in mental health . as a result data often combines all nurses .
this made it difficult to discern the unique state of canadian pmhn from that of all canadian nurses .
the first section reviews the literature from 2 sources that include a critical sociological perspective .
the next section considers the interrelation of history , gender , and the development of education for canadian pmhn through the review of 6 social , historical , and analytical studies .
the final section focuses on the quality of canadian mental health psychiatric nurses work life from 6 survey reports .
wall utilized a sociological outlook , as described in section 2.3 , to critique nursing practice settings and nursing research .
the experiences of nurses can be further understood through a lens that encompasses professionalization and organizational influences .
in addition , gender is ingrained in all of nursing where a patriarchal culture manifests . according to wall ,
the control of medicine over nursing is evident in nursing 's utilization of quantitative approaches in research that is consistent with the history of medicine .
health care , being a largely institutionalized entity , has its own way of socializing nursing and continues to place medicine at the apex of health care despite health care reformation .
conversely , wall does not comment on the socialization or culture of medicine with regards to gender , as medicine being once an overwhelmingly male profession has increased the number of female practitioners .
furthermore , the discussion by wall discusses nursing in general and does not specify particular sectors of nursing such as nps . nevertheless , a critical sociological paradigm may help to further understand issues in nursing and pmhn related to knowledge , gender , professionalization , and organizations .
utilized smith 's sociological frame of institutional ethnography to reconsider the stress in nursing through interviews , focus groups , and observation of pediatric intensive care nurses [ 21 , 22 ] .
results suggest that stress is framed within the social structure of organizations that may have hierarchical and power - based relations . articulating patient matters
studies concerning nursing stress or vicarious trauma often neglect how gender influences the life of nurses .
theories that are utilized in nursing research may avoid gender analysis . without a gendered analysis ,
nursing may further benefit by utilizing a gender perspective in research to potentially unveil social patterns that may influence teamwork and collaboration .
gender , race , and class are aspects of nurses ' identities but may not be within the range of the theory utilized for the research .
the critical gendered and sociological perspectives as depicted by wall and mcgibbon et al . provided the lens in how pmhn is perceived in relation to the following results .
tipliski contributes to the understanding of canadian pmhn history in the study entitled parting at the crossroads : the emergence of education for psychiatric nursing in three canadian provinces ,
gendered roles may have permitted psychiatry to maintain control of psychiatric nursing education in the western provinces . unlike ontario , where nurses were able to assume control over pmhn , thereby allowing pmhn incorporation into general nursing education , the provinces of manitoba and saskatchewan permitted psychiatry to prevent the merging of pmhn with general nursing . from here , as tipliski explains , two different models for canadian pmhn education developed leading to the class of the rpn that only exists in the western provinces .
this may have fostered a partition in the pmhn nursing force in canada , as the rpn designation presents a separate regulation of nurses that is nonexistent east of manitoba .
tipliski describes how the nursing leaders of saskatchewan and manitoba failed to assert control over nursing education . in 1955 , the canadian nurses association ( cna ) recognized how the separate training that was occurring in the western provinces was not conducive to the efforts to professionalized nursing for all of canada .
unfortunately , a progressive movement by the nursing leaders of that time to bring psychiatric nursing under the umbrella of general nursing in the 1950s dissolved .
this left the western provinces with a split nursing educational approach where psychiatric nurse training remained separate from general nursing education and in the hands of psychiatrists .
although psychiatric nursing is no longer controlled by the psychiatrists in the western provinces , separate training for rpns remains . from this perspective , it can be appreciated how female nursing leaders in ontario influenced historical developments to integrate psychiatric nursing education , which was consistent with earlier efforts made by cna to converge the psychiatric nursing education with general nursing in the western provinces .
ontario nurse leaders were able to gain control over their own nursing education and practice and were aided by the government .
tipliski explains how the ontario male medical superintendents attempted to keep mental nursing separate from general nursing thus hindering the professionalization of nursing .
the contributions from nettie fidler in 1933 , a nurse graduate from the toronto general hospital , together with a report by professor george weir that recommended the merging of general with mental nursing , stimulated the progression by the registered nurses association of ontario ( rnao ) to advocate for closure of the separate schools providing only mental health training .
it is possible that this account may overlook some of the other possibilities accounting for how pmhn developed in diverse ways , yet tipliski provides a description of the history of pmhn that may help to explain how gender dynamics influenced the development of pmhn in canada . despite the variation between educational approaches that resulted in the rpn designation for the provinces east of ontario ,
there is a lack of evidence revealing how pmhn care varies given the differing education preparations .
of interest is tipliski 's reference to how gender may have been a factor in the development of the rpn designation of the western provinces .
as tipliski depicts nurses were assumed to be fitting to provide care due to their female gender with their inherent ability to nurture .
the view of female nurses as nurturers may have been a patriarchal belief held by the male medical superintendents .
tipliski also considers the patriarchal context of pmhn , where the nurse leaders of the western provinces may have struggled against the authority of the medical profession . on the other hand
, tipliski does not speak of the power issues between general nursing and medicine or the professionalization the regulated psychiatric nurses of the western provinces experienced .
dooley argues that the separate class of psychiatric nurses in manitoba developed their own unique craft that is specialized for the mentally ill population . in this study ,
the account from manitoba mental health nurses of the 1930s supports the view that the development of their nursing profession was the outcome of their cooperation with physicians .
female mental health nurses in manitoba considered themselves skilled and at a higher level than the male attendants .
the female nurses were often in supervisory positions directing the personal care given by the male attendants .
this contrasts with tipliski 's view on the separate division of psychiatric nursing being related to paternalistic structures and in which female mental nurses could not overcome male physicians and psychiatrist 's domination that sought control and power .
yet the manitoba mental health nurses that would become rpns asserted their distinct class , and this has enabled the continuation of the separate training for mental health nursing that continues to manifest inside the western provinces .
evidence to suggest that the separate divisions of psychiatric nursing education in canada that have any effect on the quality of mental health care has not been substantiated in research .
one must also acknowledge the circumstances that influenced canadian women as nursing leaders in the past .
for instance , dooley describes the social context of the interwar years where women were looking for ways to support themselves , and mental health nursing provided a way to live that would provide regular meals and housing for mental health nurses . from this description , it is possible that pmhn also developed in diverse ways in canada as a result of social and economic circumstances in addition to gender influences , that have yet to be fully explored .
hicks provides further details concerning manitoba 's adoption of the rpn model . through a genealogical analysis ,
gender stands out as a main influence in this movement where the male leaders of the psychiatric nursing associations of the adjacent western provinces were influential in drawing manitoba to call for the distinct nursing class .
the male nurses of the rpn psychiatric nursing associations in the western provinces developed a collegial relationship with the male attendants of manitoba who sought rpn status .
this presents an interesting insight with regards to gender , in that the men were able to increase their strength and power through the joining with the male psychiatric nurses of manitoba .
this also diverges form tipliski 's view where the psychiatric nurses were submissive to medical authority in relation to the female gender .
in addition , the separate psychiatric rpn distinction was favored by the male medical superintendents as there was a lack of interest by the general nurses to work in psychiatry , which may have also furthered the movement towards the rpn class .
hicks considers the large number of male attendants and the significance of gender in the creation of the rpn designation .
male attendants provided custodial care and sought to provide nursing care that would elevate the status of the male attendants . unlike dooley and tipliski who focus on the female gender of nursing , hicks depicts the collegiality and support of male rpn leaders of the western provinces .
the canadian council of psychiatric nursing provided support to the manitoba attendants who sought rpn status .
the rpn emergence may be seen as a way for the male gender to enter into nursing in a time where nursing was culturally enshrined as a female role and the attendants of psychiatric institution sought status and class through the rpn profession . in this way
, hick 's study demonstrates how male gender has influenced the history of canadian pmhn .
boschma et al . examined nurses ' stories that further the understanding of the development of pmhn in alberta .
rn status was recognized as being desirable for pmhn and could be achieved by mental health nurses by taking an extra 18 months of training in a general hospital after completing 2 years in a psychiatric hospital .
women were sought as nurses by the governing psychiatrists for their caring and compassionate nature .
the male attendants were excluded from nursing because of their gender and became increasingly resentful . the male attendants sought recognition and professional status .
like saskatchewan , the separate status from rns was lobbied for by the male attendants and in 1963 registration was given to psychiatric nurses . in this sense ,
the development of the separate rpn status stems from the gender division that favoured rn status to females leaving male mental health attendants to seek professional status by becoming rpns .
this differs from the view held by tipliski that mainly denotes the oppression of largely female nurses by the medical superintendents .
likewise , the account by hicks and boschma et al . also varies from the perspective by dooley .
dooley explains that the female psychiatric nurses saw the rpn category as separate and offering more to the mental health care of patients than the general nurse education could offer . despite the development of the rpn class , mental health care in
alberta continued to suffer and the need for further education continued pressure to achieve rn status .
professionalization through the regulation of rpns is described further by the interweaving of data from boschma 's case study of community mental health in alberta and hick 's historical review of psychiatric nursing in manitoba .
boschma explains that alberta graduates from the mental hospitals that started in the 1930s met with resistance from the general nursing organizations leading to the formation of the separate professional organization for rpns .
hicks , consistent with tipliski , indicates that the alberta association of registered nurses ( aarn ) had opposed the plan by superintendent charles baragar to establish a program to train psychiatric nurses .
the aarn recommended that general nurses may complete a postgraduate psychiatric course or combine with general nursing students during a third year of training .
baragar , however , was able to succeed in his intentions through his appeal to the minister of health , and the program was implemented .
this program included a total of 4 years of instruction at the mental hospital with 2 of these years occurring at the general hospital .
male attendants were restricted from attending and had to complete a three - year psychiatric program .
the male attendants went on to successfully lobby for the alberta psychiatric professional nursing association .
hicks explains that the association 's activities during that period may have been more union type activities rather than professional . in the 1950s , the movement to form the psychiatric nursing association was developing in manitoba .
the canadian nurses association recommended combining the rn and psychiatric training programs in disapproval of the two models emerging for psychiatric nursing in canada . despite efforts to organize this
boschma and hicks further explicate that beyond the development of the rpn profession to meet the poor staffing of the mental health facilities , the profession of psychiatric nursing in the western provinces grew to deliver a much needed service that provides specialized mental health nursing differing from what general nursing could supply .
the rpn profession may advance mental health nursing knowledge and care through the experiences gained from this profession 's unique historical development . in 2001 ,
the canadian nursing advisory committee ( cnac ) formed as a result of the recommendations by the advisory committee on health human resources ( achhr ) .
the achhr 's first recommendation of the nursing strategy for canada was to create the cnac .
the cnac included nursing representatives from the three nursing professions of rns , rpns , and lpns .
the cnac 's main goal concerned the quality of nursing work life and the identification of provincial and territorial strategies to enhance nursing work life . with the shrinking workforce
the cnac commissioned 6 projects which looked at strategies to improve workplaces , costs related to absenteeism and overtime , workload issues , satisfaction of nurses in the workplace , workplace respect , and autonomy and health care organizational structures . as a result
the recommendations can be summarized into three categories . the first category concerns management issues and resources .
there is a need to reduce nursing 's pace and intensity , increase full time work , decrease sick time and overtime , and enable full scope of practice .
the second recommendation speaks to professional work settings that foster a thriving and dedicated workforce .
respect for nurses is key to this recommendation , as well as education at the master and doctoral levels .
monitoring of the health of nurses and workplaces and disseminating information to keep nurse abreast of initiatives and education are considered .
accreditation , awards to promote quality of nursing work life , continued research on the nursing workforce , implementation of regional nursing committee recommendations , national nursing retention , and recruitment campaigns with a heightened emphasis for diverse and aboriginal groups are a few activities mentioned within this recommendation .
the recommendations strive to rectify the main issues that concern the shortage of nurses , the lack of educational opportunities , the limited scope of nursing practice , and the unfavourable working conditions , which are also applicable to the rpns and rns who provide pmhn . however , the cnac recommendations lack consideration of gender and its intersection with professionalization and workplace culture that culminate in the core of issues , including patriarchal culture- and power - based hierarchical organizations that impact nursing work lives [ 20 , 21 ] . lacking also from the cnac recommendations
, pmhn needs to organize as a united front , so that its voice is heard .
this may be challenging as more powerful stakeholders , like medicine , have traditionally dominated health care [ 20 , 21 ] .
maslove and fooks conducted a study to determine the degree of implementation of the 51 cnac recommendations made in 2002 as requested by the office of nursing policy at health canada .
policies of the stakeholder organizations were assessed to determine their impact on facilitating the implementation of the recommendations .
their methodology included scanning of websites , sending letters to 94 stakeholders to determine their progress , and interviewing 14 informants to identify barriers and supports .
the 94 stakeholders included employer organizations , the federal government , provincial / territorial governments , unions , professional associations / regulators , educators , research community , and national organizations .
findings were then shared with nursing stakeholders at a roundtable to enable feedback . the recommendation to increase the number of education seats occurred in a uniform manner . however , other recommendations occurred in some areas but not all , and there was difficulty in determining what had occurred nationwide .
implementation of the recommendations including workload measurement systems , increased full time positions , analyzing sick time , increasing nurse mentors , and flexible scheduling were not consistently taking place throughout canada .
the lack of stable funding was depicted as a barrier to implementation of the recommendation to develop secure jobs .
the varying professional associations and regulatory colleges that exist in each province may complicate assigning recommendations .
determining what organizations should be responsible for is a priority concern , and employers need support to enable improvements that will impact nursing quality of work life .
organizations may require government funding in order to implement the recommendations that will enhance nursing work environments .
together , the cnac 's recommendations and the study by maslove and fooks include data from rpns and general nurses who also provide psychiatric care in the provinces east of manitoba .
poor working conditions may negatively impact quality care , and the lack of action and accountability by organizational and provincial territorial leaders and their support to employers may adversely impact the mental health of both nurses and the canadian population , which they serve .
findings from the 2005 national survey of the work and health of nurses ( nswhn ) examined the health of canadian regulated nurses as related to their work environment .
nineteen thousand nurses inclusive of rns , rpns , and lpns were surveyed with a response rate of 80% .
of note is that 44% of males reported assault compared to 28% of female nurses .
it is not known if this points to male nurses being more likely to be assaulted or more likely to report assault .
high physical demands were reported by 75% of lpns , 60% of rns , and 45% of rpns .
gender difference was not specified for physical demands , that is , if male or female nurses reported higher physical demands .
the mental health of nurses was adversely associated with evening shifts and employment in long - term care facilities .
lack of respect and low support from coworkers and superiors were linked with poorer mental health .
in addition , the mental health of nurses was also affected by elevated job strain , low autonomy and control , and poor physician relations .
one in ten nurses reported having depression and needing to take time off in relation to their mental health .
the finding of depression in nurses is compared with the overall employed population that utilized data from the workplace and employee survey , labour force survey , and the community health survey ; however , comparisons with specific groups or professions are not explicit . of all nurses , including both male and female
the experience of depression by sex was not included for nurses but for all ; employed the 4% of men and
also at the time of this study in 2005 , one - fifth reported their mental health difficulties interfering with their jobs .
the findings of this survey may further illuminate the working conditions for nurses and the implications for their mental and physical health .
the health policy research bulletin ( hprb ) is published usually twice yearly by health canada with the purpose of reinforcing the evidence that supports decision making in health policy . in 2007 ,
the hprb 's issue , titled the working conditions of nurses : confronting the challenge , focused on the canadian nurses ' working conditions and implications for the country 's health care system . between 1997 and 2005
overtime by rpns , lpns , and rns increased in all areas where nurses work by 58% . in light of the high overtime rates
, it is questionable if the current level of full - time nursing positions in mental health care in canada is sufficient to adequately care for those with mental illness .
all areas in nursing face similar pressures concerning increased overtime and a need for more full time work ; however , mental health settings entail frequent interactions with challenging and difficult behaviours .
this may intensify the stress on nurses working in mental health care where there is shortage of full - time nurses .
a survey of 1015 rpns in manitoba was conducted to determine the predictors , prevalence , correlates , and distribution of vicarious trauma and burnout .
the survey included the maslach burnout inventory , the traumatic stress institute belief scale , and a section on posttraumatic stress disorder ( ptsd ) symptoms .
emotional exhaustion was the highest in rpns working in community services and acute care hospitals .
constant interruptions , burdensome responsibility , increased trauma work , depersonalization , and elevated vicarious trauma scores were linked with higher emotional exhaustion levels . with regards to vicarious trauma , 21% had persistent thoughts in relation to client trauma , and 30% experienced a heightened level of arousal .
client trauma is the exposure to a stressful experience that overwhelms a person 's coping mechanism .
fifty - five percent of those involved in client trauma met one criteria of ptsd , and 48% responded that symptoms were troublesome to some degree .
lack of peer support and skills to deal with trauma could be rectified by increased education and team building .
the rpns in this study also reported a high level of personal accomplishment , which is associated with low burnout .
personal accomplishment includes the perception that clients are improving and the rpns have the skills necessary to help individuals with mental health disorders .
the study is significant in that it mirrors the results of the studies in the preceding discussion concerning nursing quality of work life .
stress is evident in nurses working in mental health care and impacts the mental health of the caregivers . on the other hand ,
seven focus groups with rpns from a diversity of health care settings took place in manitoba over a nine - month period .
the themes that emerged consisted of changes from institutional care to the community , variations in professional position , primary and secondary care and prevention , lack of provincial communication , and consistency amongst policies .
this change led to an extension of their roles in many different settings including emergency departments , treatment centers , and acute psychiatry .
rpn professional status has required a move to more autonomous roles within the primary care setting where professional competence is emphasized .
emphasis on health promotion and prevention is in contrast to the traditional approaches that focused on treating illnesses in institutionalized settings .
study participants described lack of consistency with mental health standards , protocols , and policies in the region .
in the same study , the rpns became more familiar with the growing emphasis client - centered care and the need for mental health consumers to be involved in the decision making process .
the transition from the traditional institutionalized care where decisions were made for the clients had left some rpns feeling unprepared and concerned that the client may not choose what is best for them .
the importance of engaging the family has become more apparent and this contrasts to the way care was provided in the past where family involvement was limited in the institutional setting .
the education of rpns and rns is essential to meet the changing approaches to caring for those with mental illness and the growing recognition of the impact of social determinants of health on the health and well - being of diverse populations .
there is scant research that clearly indicates how the diverse educational preparation of nurses working in psychiatric care impacts the quality of care for persons with mental health disorders .
given the burden of mental illness in canada there is need for more research that analyzes the psychiatric educational preparation of nurses in relation to accessibility to mental health care and therapeutic and treatment outcomes .
client - centered care requires advocacy and involvement with family and community beyond the confines of institutional care and this may require increased education specific to the changing dynamics of mental health care within the community setting . according to ryan - nicholls ,
, participants in the focus groups discussed concern about the 2-year diploma program changing into the 4-year baccalaureate degree for rpns , graduating only 15 students per year as compared to 60 students per year from the diploma program .
therefore , the issues for rpns parallel to the issues of the broader nursing workforce , where the shift to higher education has impacted the size of the nursing workforce , but at the same time the nursing graduates of today have increased knowledge to meet the current challenges of health care .
the diversity between the organization of pmhn and education between the western provinces and the rest of canada has connections with events of the past and intersects with gender , professionalization , and the predominating organizational culture .
the historical analyses of nursing may foster additional inquiries that may benefit nursing knowledge through learning from past approaches and gaining new perspectives .
overall pmhn in canada is challenged to further itself to meet the lack of accessible mental health services .
there is evidence that nurses are stressed , and there is a need to enhance a coordinated national approach for advanced degrees in pmhn .
the lack of a uniform approach to pmhn education in canada has consequences for the further development of pmhn and may generate barriers to further pmhn to meet the growing mental health care challenges .
the implementation of standards by the canadian federation of mental health nursing is brought to the discussion here in order to explain how standards are an important way to address the diverse forms of pmhn education .
the standards foster an overarching educational approach for pmhn that enables quality pmhn practice . in 2006 , the third edition of the canadian standards for psychiatric - mental health nursing was released in an effort to prompt nurses to adopt the standards into daily practice and further nursing reflection on their work .
the latest standards were developed after consultation with canadian consumers of mental health across canada .
acknowledge that systemic issues , that is , labeling , stigma , caregiver , and treatment role , affect pmhn but emphasize the need for nurses to know their clientele to foster therapeutic relationships .
systemic factors including workforce size , workload , violence in the workplace , nursing scope of practice , and accessibility to pmhn may have important implications for pmhn and their daily practice .
however , systemic factors must be addressed to foster pmhn 's delivery of high quality care that are consistent with the standards for psychiatric - mental health nursing .
furthermore , as an influential and strong stakeholder that can affect change in mental health care , pmhn may benefit from a uniform educational process throughout canada . also of concern
is the mental health of nurses who experience high stress . as nurses are mostly women and nurses form the largest group of health care providers , the ramifications for the health and productivity of the canadian society are especially disconcerting if the majority of nurses are experiencing reduced quality of work life . as wall and mcgibbon et al .
point out , a sociological paradigm enables the discussion of sensitive issues including the gendered impact over nursing and the need for research from a perspective that views critically the influence of gender on quality of nursing work life .
the labour divisions in organizations where nurses practice are entrenched in hierarchal power struggles that undermine nursing knowledge and autonomy and contribute to poor quality of work life and stress .
although several studies were found in relation to the history of pmhn for the western provinces and ontario , no studies were located that convey the full pmhn history of eastern canada . while there were government documents concerning the quality of work life for all nurses , it was difficult to abstract information specific to pmhn from these documents .
for instance , although the nswhn found a high incidence of depression amongst nurses , the percentage of nurses in pmhn experiencing depression was not given .
it is acknowledged that the whole puzzle of what pmhn is like in canada is not complete .
furthermore , as nps are becoming more established within the canadian health care system there is a need for increased research that reveals np mental health care initiatives and activities . at present
, there is a lack of research pertaining to canadian nps employed in mental health and their quality of work life . despite this ,
the findings reported here depict important points regarding the issues that concern all of pmhn and how they impact the provision of mental health care for canadians .
while nps have made significant progress in achieving prescriptive authority in the area of primary care , minimal movement has occurred regarding the feasibility of nps specializing in mental health . in canada
, there are no educational programs or legislated provisions for nps who wish to specialize as a psychiatric np or who already have extensive experience providing mental health care . the existing situation for nps practicing within mental health care settings
is hampered by the absence of recognition for the psychiatric mental health nurse practitioner ( pmhnp ) in canada .
prescriptive authority can only be obtained in ontario through registration in the extended class in the designated specialties , np paediatrics , np - primary health care and np - adult .
the educational programs that prepare nps for these specialties focus on the medical and physiological aspects of the specialties with limited content on mental health . in 2011 ,
the nursing act , 1991 , no longer stipulates the prescribing of only listed medications by nps . with the exception of medications under the controlled drugs and substances
act , nps are now able to prescribe medications commonly used in mental health care .
this allows nps to further their care for clients needing mental health care ; however , it may also point to the need for comprehensive mental health training for nps .
it beckons further exploration if the existing mental health nursing programs offered throughout canada could enable the development of pmhnp programs that would enable rns or rpns and nps to obtain the competencies of the pmhnp .
communities with insufficient mental health care resources may be better served by nurses with an expanded scope of practice with specialized mental health education . in addition , although there have been changes to legislation , specifically regulation 965 of the public hospitals act , that gives admitting and discharging privileges to nps ; changes to the mental health act have not been made and regulated forms enabling admission to a hospital for psychiatric assessment can not be completed by canadian nps . when the np is the person 's main care provider , it would seem prudent that the np should be involved in decisions concerning psychiatric admission and discharge .
the variability of pmhn in canada presents both challenges and opportunities to the advancement of pmhn education .
given the challenge for increased accessibility to mental health care services and treatments , the development of already existing nursing programs in canada could bond by striving to achieve the same psychiatric educational nursing standards .
to further mental health care it may be possible for all nursing programs to uphold the same standards for psychiatric nursing education .
the cna motioned a resolution in 2005 to include rpns as it was recognized that a separate national level for rpns would hinder professional nursing practice and the power for canadian nurses to advocate for change . amending the division between rpn and rns and to enable certification and eligibility of cna membership for both groups may strengthen the canadian pmhn workforce .
gallop considers how the education system in canada lacks prospects for nurses working in mental health to obtain advanced degrees in pmhn , although , as previously mentioned , there is new potential for rpns and nurses to obtain master degrees specializing in mental health nursing west of ontario . as mental health care is no longer confined to institutional settings and mental health training is pertinent to all areas of health care , the necessity for advanced mental health education is relevant to all health care settings where nurses practice .
nursing needs to facilitate advanced education in mental health so that people within the primary care setting also benefit from the knowledge and expertise of nurses who have additional mental health education .
bridging programs or additional educational opportunities for psychiatric nurses wishing to broaden their knowledge specifically to prescribe psychiatric treatments or medications may be helpful .
partnerships between provincial nursing bodies to foster national standards for pmhn education and bridging programs to a pmhnp program if developed may represent new opportunities for all canadian nurses . the report entitled
2009 - 2010 , conducted by the canadian nurses association ( cna ) and the canadian association of schools of nursing ( casn ) , reveals an increase of 64.3% from 2009 to 2010 for graduates for doctoral programs . in 2010 , 47.6% of nps had a master 's , and 0.8% of np had a doctorate degree .
more nurses and nps are acquiring advanced degrees . with this trend for advanced education ,
it is likely that mental health education for all nurses and nursing research pertaining to mental health and pmhn will flourish .
pmhn in canada must take action to meet the goals as set out by the mental health commission of canada .
national standards for psychiatric nursing education for all canadian nursing education programs may positively impact mental health care .
advanced education for pmhn including the development of a canadian pmhnp program may further the accessibility of psychiatric treatments .
psychiatric mental health education for all nurses will complement primary health care and the provision of mental health care in general hospitals and community settings .
efforts to unite and form partnerships with the varying groups providing pmhn at a national level may empower pmhn as a powerful stakeholder in mental health care reform .
also , the mental health of nurses in light of their quality of work life is a red flag for all leaders in the canadian health care system . there is a need to understand the circumstances of nurses with regards to occupational stress and barriers to advanced education in mental health nursing .
although the full picture of what canadian mental health nursing looks like today can not be fully realized by this synthesis , important issues facing pmhn have been identified through the analysis of history and application of a critical and gendered sociological lens .
another chapter on the evolution of canadian pmhn has yet to be written ; hopefully , it will entail how pmhn works together with all stakeholders equally to provide the best care possible . | a society that values mental health and helps people live enjoyable and meaningful lives is a clear aspiration echoed throughout our canadian health care system .
the mental health commission of canada has put forth a framework for a mental health strategy with goals that reflect the virtue of optimal mental health for all canadians ( mental health commission canada , 2009 ) .
canadian nurses , the largest group of health care workers , have a vital role in achieving these goals . in canada ,
two - thirds of those who experience mental health problems do not receive mental health services ( statistics canada , 2003 ) . through a gendered ,
critical , and sociological perspective the goal of this paper is to further understand how the past has shaped the present state of psychiatric mental health nursing ( pmhn ) .
this integrative literature review offers a depiction of canadian pmhn in light of the intersections of history , gender , education , and quality of nursing work life .
fourteen articles were selected , which provide a partial
reflection of contemporary canadian pmhn .
findings include the association between gender and professional status , inconsistencies in psychiatric nursing education , and the limitations for canadian nurse practitioners to advance the role of the psychiatric mental health nurse practitioner . |
sertoli - leydig cell tumours ( sclts ) are rare sex - cord stromal tumors of the ovary .
retiform sclt 's are rare histological variant of sclt with an average age of presentation being 16 years . to the best of our knowledge
the retiform variant of sertoli leydig tumors reported so far are seen in less than 10 years of age .
we present a rare case of retiform variant of sclt 's presenting in a 42-year - old female .
a 42-year - old female presented with painless lump in the lower abdomen for 7 years .
largest measured 14 12 cm , smallest measured 3 3 cm . on cutting fleshy , firm tissue was found within the cyst wall [ figure 1 ] .
microscopic examination showed ovarian tumor with tubular , cord like pattern lined by bland cuboidal to columnar cells .
also seen is retiform pattern at few places and scattered cells with abundant eosinophilic cytoplasm suggestive of leydig cells .
leydig cells showed focal positivity for vimentin and negativity for leydig cells [ figures 7 and 8 ] .
histological diagnosis of retiform variant of sertoli leydig cell tumor of ovary stage 1 ( t1n0m0 ) was made .
the patient was followed up to 2 years , she did not have any complaints .
gross appearance of sertoli leydig cell tumor sertoli cells arranged in cords , sheets and aggregates ( he 100 ) retiform pattern in serrtoli- leydig tumor ( he 100 ) sertoliform cells with clear cytoplasm ( he 400 ) leydig cells showing eosinophilic cytoplasm ( he 100 ) sertoli cells showing atypia ( he 400 ) cytokeratin positivity in sertoli component of sertoli leydig cell tumor of ovary ( 100 ) vimentin positivity in leydig component of sertoli leydig cell tumor of ovary ( he 100 )
however , retiform sertoli - leydig cell tumors present in the average age of 16-year - old . clinically ,
non - virilised patients present with non - specific symptoms like abdominal mass , pelvic pain .
it is seen that 80% of the patients with ovarian slcts and virilising manifestations present with elevated serum levels of testosterone and androstenedione .
however , sclts are unilateral and are confined to ovary at the time of diagnosis .
computerised tomography ( ct ) , magnetic resonance imaging ( mri ) , and positron imaging tomography ( pet ) scans can provide us a better visualisation of the extra - ovarian or metastatic spread of the tumor .
well - differentiated slct have an average diameter of 5 cm whereas intermediate type and poorly differentiated types have an average diameter of 15 cm .
microscopically , they are divided into following categories :
well - differentiated or meyer type 1 : they constitute 11% of slct.intermediate type or meyer type 2 : they constitute 54% of slct.poorly differentiated ( sarcomatoid or undifferentiated or meyer type 3 ] : they constitute 13% of slct.sclt with heterologous elements like skeletal muscle , cartilage.retiform type ( 15% ) : they resemble rete of ovary or testes .
staging wise t1 means tumor limited to the ovaries , t2 means tumor involves one or both ovaries with pelvic extention , t3 means involvement of one or both ovaries with microscopically confirmed peritoneal metastasis outside the pelvis .
intermediate type or meyer type 2 : they constitute 54% of slct . poorly differentiated ( sarcomatoid or undifferentiated or meyer type 3 ] : they constitute 13% of slct .
staging wise t1 means tumor limited to the ovaries , t2 means tumor involves one or both ovaries with pelvic extention , t3 means involvement of one or both ovaries with microscopically confirmed peritoneal metastasis outside the pelvis .
a total abdominal hysterectomy is considered as the treatment of choice in patients with unfavourable findings like rupture , extra - ovarian spread , poorly differentiated neoplasm , heterologous mesenchymal differentiation .
post - operative chemotherapy , radiotherapy or a combination of both , may also be considered in the patients with above mentioned poor prognostic factors .
similarly the 5 year survival rates for stage 1 tumor is 95% and that for stage iii and stage iv is zero percent . | sertoli - leydig cell tumors are the uncommon sex - cord stromal tumors of the ovary .
we report a case of 42-year - old female with retiform variant of sertoli - leydig cell tumour .
she presented with the complaint of mass in abdomen for 7 years .
ultrasound revealed bilateral ovarian mass suggestive of malignancy .
bilateral salpingo - oopherectomy with surgical staging was done .
the tumor was diagnosed as stage i retiform variant of sertoli - leydig cell tumor on histopathology and immunohistochemistry . |
coronary artery disease ( cad ) is one of the most common causes of death in the developed world .
the high prevalence of cardiac risk factors and associated morbidity have been reported in the iranian adult population .
mets is a growing health problem in iranian adults due to the alterations in lifestyle , low physical activity , the epidemic of obesity and insulin resistance ( ir ) [ 2 , 3 ] .
mets includes several cardiometabolic risk factors and is characterized by four essential components including intra - abdominal obesity , dyslipidemia , hypertension and impaired glucose tolerance [ 2 , 4 ] , and linked to a high risk of both type 2 diabetes and cad , and increased risk of cardiovascular events [ 2 , 5 , 6 ] .
the epidemiologic studies on mets in iran have revealed the high prevalence of mets in the north of iran ( 37.9% ) , zahedan , southeast of iran ( 28.5% ) , and in the province of zanjan , the west of iran ( 23.7% ) . based on the tehran lipid and glucose study ,
the prevalence of mets in the iranian adults was reported to be 33.7% ( 24% for men vs. 42% for women ) .
the association between mets and its components with cad has been reported in several studies using different techniques [ 1 , 5 , 6 , 10 ] . according to atp iii criteria , diagnosis of mets
is based on the presence of three or more of the five criteria including waist circumference ( wc ) > 102 in men and > 88 in women , high blood pressure ( bp 130/85 ) , high triglyceride ( tg 150 ) , high fasting blood sugar ( fbs 110 ) , and low high - density lipoprotein ( hdl < 40 in men and < 50 in women ) .
the american heart association ( aha ) and national health lung and blood institute ( nhlbi ) modified the atp iii criteria with a reduction in fbs from 110 to 100 mg / dl [ 3 , 12 ] . as well , the iranian national committee of obesity ( inco ) considered cut - offs of 95 cm for both men and women in iran , which could predict the incidence of cardiovascular disease ( cvd ) [ 3 , 13 ] .
each of mets components is considered as an independent cardiac risk factor and increased number of components elevates the severity of cvd .
central fat distribution is a more atherogenic property than peripheral obesity , and wc is evaluated to identify the central obesity .
thus , abdominal obesity is considered as one of the components of mets , which is linked to the occurrence of dyslipidemia , diabetes and risk of cardiovascular events .
ir is also one of the major characteristics of mets , and considerably affects the development of cad [ 5 , 10 ] . in a study , low hdl , high fasting plasma glucose ( high fpg ) , high bp and high tg
based on the above - mentioned evidence , diagnosis of mets and its components in patients with cad can influence prevention of disease and management programs [ 5 , 6 , 10 ] .
thus , this study aimed to investigate the prevalence of mets and its individual components in cad patients who underwent elective coronary angiography .
this cross - sectional study was performed on 200 cad patients ( mean age 58.3 11.1 years ) admitted to cardiology department of imam ali hospital in zahedan , southeast of iran , and diagnosed by a hospital cardiologist , during a 5-month period from june to october 2016 .
cad was diagnosed based on a combination of previous medical history , clinical findings ( e.g. , dyspnea , and excessive fatigue , and suspected ischemia ) , and electrocardiogram ( ecg ) changes , and confirmed with 50% or more coronary artery stenosis in at least one major coronary artery as assessed by coronary angiography .
the patients with chronic heart failure , myocardial infarction or unstable angina pectoris and acute coronary syndrome ( acs ) who needed emergent coronary intervention or surgery , hepatic and kidney diseases , hyperthyroidism , pregnancy , and patients with ejection fraction ( ef ) < 60% were excluded from study .
all patients gave oral informed consent , and ethical approval was obtained from ethics committee of zahedan university of medical sciences ( approval date april 24 , 2016 , no .
the demographic data including age , sex , physical activity , and previous medical history and medications were completed at the first meeting with patients .
physical activity was classified into three categories : no activity < 3 , and 30 min walking for at least three times a week .
the weight and height of patients were recorded with light clothes and without shoes by a seca scale ( germany ) to the nearest 0.5 kg and 0.5 cm , respectively .
body mass index ( bmi ) was calculated as weight ( kg)/height ( m ) .
wc was measured in the standing position using an inelastic measuring tape to the nearest 0.5 cm between the lower border of the rib cage and the iliac crest .
the measurement was performed on the right arm using a digital manometer ( model alp k2 , k2 - 231 , japan ) .
two recordings were carried out , and the mean levels of bp were used for analysis .
serum levels of total cholesterol , high - density lipoprotein - cholesterol ( hdl - c ) , low - density lipoprotein- cholesterol ( ldl - c ) , tg and fasting blood glucose ( fbg ) were assayed by enzymatic procedures ( pars azmon kit , tehran , iran ) using an auto - analyzer ( hitachi , japan ) . in this study , mets was described as the presence of 3 of the following criteria , according to the modified protocol of adult treatment panel ( atp iii ) , with regional cut - off value of wc 95 cm for both gender , increased fbg 100 mg / dl , hypertriglyceridemia ( tg 150 mg / dl ) , decreased hdl - c ( < 40 mg / dl in men or < 50 mg / dl in women ) , and hypertension ( systolic blood pressure ( sbp ) 130 mm hg and diastolic blood pressure ( dbp ) 85 mm hg ) .
statistical analyses were performed using statistical package for the social sciences version 21 ( spss inc . ,
data were presented as mean standard deviation ( sd ) , frequencies and percentages .
odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated for mets and individual components of it .
multivariable linear regression model , adjusted for sex , was used to evaluate the association between variables .
statistical analyses were performed using statistical package for the social sciences version 21 ( spss inc . ,
data were presented as mean standard deviation ( sd ) , frequencies and percentages .
odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated for mets and individual components of it .
multivariable linear regression model , adjusted for sex , was used to evaluate the association between variables .
the demographic and clinical characteristics of the 200 cad patients ( 82 men and 118 women ; aged 24 - 81 years ) with and without mets are presented in table 1 . of these , 49.5% had mets ( n = 99 , 95% ci : 4.8 - 9.3 ) , and it was 55.9% among women and 40.2% among men ( p < 0.05 ) .
the highest prevalence was present in patients aged > 50 years ( 87% , p < 0.05 ) . the mean wc , bmi and bp ( p < 0.0001 ) , serum levels of fbg and cholesterol ( p < 0.001 ) , tg ( p < 0.0001 ) and ldl - c ( p < 0.001 ) in cad patients with mets were significantly higher , and hdl - c levels ( p
data are expressed as mean sd . mets : metabolic syndrome ; bmi : body mass index ; wc : waist circumference ; bp : blood pressure ; fbg : fasting blood glucose ; tg : triglyceride ; ldl - c : low - density lipoprotein - cholesterol ; hdl - c : high - density lipoprotein- cholesterol .
as shown in table 2 , low hdl - c ( 84.8% vs. 67.3% , p < 0.01 ) , high fbg ( 77.8% vs. 29.7% , p < 0.0001 ) , and high wc ( 75.8% vs. 9.9% , p <
0.0001 ) were the most prevalent risk factors in cad patients with mets as compared to those without mets . as well
, the findings revealed that the ors for main components of mets in cad patients with mets were as follows : low hdl - c ( or : 12.65 ; 95% ci : 5.3 - 20 ) , high wc ( or : 6.7 ; 95% ci : 3.7 - 12.1 ) , high fbg ( or : 6.7 ; 95% ci : 5.9 - 8.76 ) , high tg ( or : 2.26 ; 95% ci : 1.1 - 3.48 ) , and high bp ( or : 1.35 ; 95% ci : 1.02 - 1.8 ) .
wc : waist circumference ; bp : blood pressure ; fbg : fasting blood glucose ; tg : triglyceride ; hdl : high - density lipoprotein ; mets : metabolic syndrome ; or ( 95% ci ) : odds ratio ( 95% confidence interval )
. table 3 demonstrates that after adjusting for sex , based on multivariable linear regression analysis , the high wc ( = 0.63 , p < 0.0001 ) , high bp ( = 0.19 , p < 0.05 ) , high fbg ( = 0.14 , p < 0.01 ) , high tg ( = 0.19 , p < 0.01 ) and low hdl - c ( = 0.20 , p <
mets : metabolic syndrome ; bmi : body mass index ; wc : waist circumference ; bp : blood pressure ; fbg : fasting blood glucose ; tg : triglyceride ; ldl : low - density lipoprotein ; hdl : high - density lipoprotein .
the studies performed regarding the prevalence of mets and/or associated risk factors in cad patients have demonstrated that the rate of mets in patients hospitalized for acs was between 43% and 51% . in a similar study , in which conventional angiography was performed in suspected cad patients , 40.5% of patients had mets based on the aha / nhlbi criteria .
the findings of this study demonstrate that the prevalence of mets in cad patients is relatively higher than other regions of the world .
these differences could be due to definition of mets by different criteria , cultural mores , lifestyle - related factors such as improper food habits , physical inactivity , stress and sample size in various investigations .
different combinations of the components of mets have various effects on cvd ; however , each component acts as an independent risk factor for cad , and all of them interact synergistically , and thus lead to increased risk of cad .
the prevalence of individual components of mets with various rates in cad patients has been reported in earlier studies [ 1 , 2 , 17 , 19 , 20 ] . the abdominal obesity , dyslipidemia ( high tg and low hdl - c ) [ 1 , 2 , 21 ] , hypertension and hyperglycemia [ 2 , 21 ] most often were reported as the ms components , and the severity of cad increased with the number of components . a study performed in shanghai showed that among individual components of mets , low hdl , high fbg , and high bp had the highest or for coronary heart disease .
the results of a study in korean population showed that mets was independently linked to coronary parameters including obstructive plaque and coronary artery calcium score in the non - diabetic cad patients . among the individual components of mets ,
low hdl - c levels were markedly associated with cad in these patients [ 10 , 22 ] .
a study in tehran revealed that 78% of men and 80% of women > 20 years old had at least one risk factor of cvd .
the prevalence of high fbg , high bp , obesity , high total cholesterol , low hdl - c , and high tg was reported to be 9.8% , 20.4% , 14.4% , 19.3% , 32% , and 5.3% , respectively . in the current study ,
the cad patients with mets had significantly higher levels of bmi , wc , bp , serum lipid profile , and fbg , and lower hdl - c levels ; however , regression analysis revealed that the rate of different components of mets , including singularly a much higher rate of low hdl - c , high blood glucose and high wc , with the highest or , and a relatively high prevalence of high tg and high bp was found in patients with mets than those without mets .
several studies have demonstrated that dyslipidemia is common in iranian population [ 24 , 25 ] .
the low hdl - c was presented as one of the most prevalent abnormalities and the strongest factor that independently linked to cad in iranian population and other countries [ 10 , 22 ] .
some studies have shown that among the individual components of mets , high level glucose was markedly associated with cad .
a study reported that the prevalence of chd in the diabetic patients with mets was significantly higher than in those without mets .
the high prevalence of diabetes in the iranian population with established cad [ 26 , 28 ] , and patients undergoing elective coronary artery bypass grafts ( cabg ) was reported in some studies .
other investigation also reported that the long - term diabetes is linked to development of atherosclerosis .
it has been clarified that hypertension as one of the components of mets is increasing among the iranian population [ 28 , 31 ] , in particular , in patients with confirmed cad [ 25 , 26 , 32 ] .
several cross - sectional investigations performed in iran have shown that the incidence rate of hypertension was significantly higher in cad patients compared to healthy population [ 25 , 32 ] and in non - insulin - treated than insulin - treated type 2 diabetes mellitus .
it was higher in men than in women , and increased with older age [ 21 , 33 ] .
there is evidence that the presence of obesity / abdominal obesity and its relationship with cad is more common than other components of mets , and abdominal obesity has been identified as a central component of mets , which has a strong relationship with hypertension , ir and dyslipidemia [ 2 , 3 , 31 ] .
the coronary angiography findings in cad patients in isfahan also showed the patients who had more wc were at higher risk of heart disease . in our study , the mean of both bmi and wc in patients with mets was significantly higher than those without mets ; however , after adjusting of sex , based on multivariable linear regression analysis , a positive significant association was found only between wc and mets , suggesting that the fat distribution in the abdomen is more atherogenic than peripheral obesity and may predict the risk of cvd .
the findings of our study showed that three of mets components including low hdl - c , high blood glucose and abdominal obesity , were the strongest predictors of cad ; however , older age , physical inactivity and female gender had also predictive value . although after adjusting of sex , there was no statistically significant relationship between the categories of age and physical activity with the mets in the cad patients ; however , these variables were significantly higher among cad patients with mets than in those without mets .
similar studies in iranian adult population reported that all major components of mets increase with aging [ 19 , 35 , 36 ] , and a higher rate of mets [ 7 , 8 , 19 ] and its components has been reported in women compared with men in earlier studies .
karimi et al demonstrated that the patients with cad were more male with older age .
won et al reported that us population over 50 years of age with mets had higher chd prevalence when compared to those without mets .
it is clear that the fat tissue in women is higher than men and increases with aging . on the other hand
, inactivity reduces energy expenditure and causes weight gain , and thus leads to ir and increased blood glucose and dyslipidemia . according to the above - mentioned evidence ,
older age and inactivity are important risk factors for development of mets among cad patients .
it seems that modification in food pattern and lifestyle among iranian population leads to low activity and increases abdominal obesity , resulting in an increase in the rate of all coronary risk factors .
since many of those at risk are not identified , further prospective studies are recommended for more clarification .
a limitation of our study was that the food habits and diet intakes in our studied population were no assessed .
however , this study was performed for the first time in zahedan ( center of sistan and baluchistan province ) in southeast of iran , thus , it is the power of this research .
the results of the study showed that the dyslipidemia , abdominal obesity and hyperglycemia are more common risk factors for cad ; however , the combination of all components of mets may be a secondary contributor to cad risk .
the results of the study showed that the dyslipidemia , abdominal obesity and hyperglycemia are more common risk factors for cad ; however , the combination of all components of mets may be a secondary contributor to cad risk .
this work was supported by the research deputy of zahedan university of medical sciences , zahedan , iran .
interpretation of data : farzaneh montazerifar , mansour karajibani , ahmad bolouri , and milad mahmoodi mozaffar .
revision of the final manuscript : fazaneh montazerifar , mansour karajibani , and ahmad bolouri . | backgroundmetabolic syndrome ( mets ) is a worldwide health problem , which is growing in iranian adults .
mets is associated with risk of type 2 diabetes and coronary artery disease ( cad ) . in this study
, we aimed to investigate the prevalence of mets and its individual components in cad patients.methodsthis cross - sectional study was performed on 200 cad patients who had undergone elective coronary angiography at the cardiology department .
anthropometric indices including waist circumference ( wc ) and body mass index were measured .
blood samples were obtained to determine glucose and lipid profile .
mets components were defined according to the modified adult treatment panel iii ( atp iii ) criteria.resultsthe prevalence of mets among patients was 49.5% ( women : 55.9% ; men : 40.2% ; p < 0.05 ) . the prevalence increased with age . the low high - density lipoprotein - cholesterol ( low hdl - c ) ( 84.8% ) , high fasting blood glucose ( high fbg ) ( 77.8% ) and high wc ( 75.8% ) were the most prevalent risk factors in cad patients with mets.conclusionsrecent data indicate that the dyslipidemia , hyperglycemia and abdominal obesity are crucial predictors of mets in cad patients .
further prospective studies are recommended for more clarification . |
in all domains of life , nucleotide - based second messengers allow a rapid integration of external and internal signals into fine - tuned regulatory pathways that control cellular responses to changing conditions . as a unifying theme , a basic second messenger control module consists of two distinct enzymes for synthesis and degradation of the second messenger and a nucleotide sensor that , upon ligand binding , interacts with a target to produce a cellular output ( hengge , 2009 ) .
3 , 5-cyclic diguanylic acid ( c - di - gmp ) , which is not produced in archaea or eukaryotes , was first discovered as an allosteric effector of cellulose synthase in gluconacetobacter xylinus and is now recognized as one of the most important and widespread second messengers in bacteria .
c - di - gmp is synthesized from two molecules of gtp by diguanylate cyclases ( dgcs ) , which are characterized by active site ggdef motifs ( a - site ) ( paul et al . , 2004 ; chan et al . ,
the majority of active dgcs also carry a so - called inhibitory or i - site motif , rxxd , which is involved in feedback inhibition ( christen et al .
, 2006 ; schirmer and jenal , 2009 ) . specific phosphodiesterases ( pdes ) , which harbor eal or hd - gyp domains , degrade the cyclic dinucleotide ( schmidt et al . , 2005 ; christen et al . , 2005 ;
the enzymatically active domains involved in c - di - gmp turnover are often associated with diverse sensory domains , thus enabling cells to adjust second messenger levels in response to different environmental stimuli ( hengge , 2009 ) .
the binding of c - di - gmp to effector proteins impacts diverse processes such as adhesion , virulence , motility , and biofilm formation in unicellular , flagellated bacteria ( rmling et al . ,
the known c - di - gmp - binding motifs of these proteins are limited but include degenerate ggdef domain proteins carrying i - site motifs ( duerig et al . , 2009 ;
, 2007b ; petters et al . , 2012 ) , inactive eal domain receptors ( navarro et al . , 2009 ;
2009 ) , and pilz domain - containing proteins ( amikam and galperin , 2006 ) .
transcription factors that sense c - di - gmp lack these common c - di - gmp - binding motifs and thus must be identified experimentally .
the sparse list of known c - di - gmp - responsive transcriptional regulators includes the tetr - like activator ltma from mycobacterium smegmatis ( li and he , 2012 ) , the crp - fnr - like transcription factor clp from xanthomonas ( chin et al . , 2010 ; leduc and roberts , 2009 ) , bcam1349 from burkholderia ( fazli et al . , 2011
) , the ntrc - type protein fleq from pseudomonas aeruginosa ( baraquet and harwood , 2013 ) , and vpsr from vibrio cholerae ( srivastava et al . , 2011 ) .
the only c - di - gmp - responsive transcription factor for which structural information is available and hence c - di - gmp binding is understood is vpst , which is a member of the well - studied fixj - luxr - csgd family of response regulators .
the vpst structure revealed a characteristic response regulator fold and a w(f / l / m)(t / s)r c - di - gmp - binding motif ( krasteva et al . , 2010 ) .
notably , in all known structures of c - di - gmp - binding effector proteins or enzymes , the c - di - gmp is bound either as a monomer or intercalated dimer .
biophysical studies suggest the possibility of higher order oligomeric forms of c - di - gmp , but they have yet to be observed in any biological context ( gentner et al . , 2012 ) .
while the roles played by c - di - gmp in controlling cellular processes in unicellular bacteria are becoming clear , the function(s ) of c - di - gmp in multicellular , nonmotile bacteria such as streptomyces are unknown .
the complex streptomyces life cycle involves two distinct filamentous cell forms : the growing or vegetative hyphae and the reproductive or aerial hyphae , which differentiate into exospores for dispersion through a massive synchronous septation event ( flrdh and buttner , 2009 ) . in the model species streptomyces venezuelae
, there are three ggdef proteins , two proteins with hd - gyp domains , and five proteins containing both a ggdef and an eal domain ( figure 1a ) . altered expression of the ggdef proteins , cdga and cdgb , and deletions of the eal proteins , rmda and rmdb , have a significant impact on streptomyces growth progression , suggesting that c - di - gmp plays a role in controlling developmental processes in multicellular bacteria ( den hengst et al . , 2010 ;
interestingly , cdga and cdgb have recently been identified as direct regulatory targets of the developmental master regulator bldd ( den hengst et al . , 2010 ; tran et al . ,
mutations in the bld loci block the formation of aerial hyphae , resulting in a bald
phenotype , and also affect the production of antibiotics ( mccormick and flrdh , 2012 ) .
bldd sits at the top of the regulatory cascade controlling development , serving to repress expression of sporulation genes during vegetative growth ( den hengst et al . , 2010 ) .
in streptomyces coelicolor , bldd controls the expression of at least 167 genes , including 42 genes ( 25% of the regulon ) that encode regulatory proteins ( elliot et al . , 2001 ; den hengst et al . ,
are many genes known to play critical roles in streptomyces development , including other bld regulators ( e.g. , blda , bldc , bldh / adpa , bldm , and bldn ) , several whi ( white ) regulators required for the differentiation of aerial hyphae into spores ( e.g. , whig and whib ) , and genes encoding critical components of the cell division and chromosome segregation machineries such as ftsz , ssga , ssgb , and the dna translocase sffa ( den hengst et al . , 2010 ; mccormick , 2009 ) .
we show that bldd is a c - di - gmp - binding effector protein , thus revealing a link between c - di - gmp signaling and the development of multicellular bacteria .
specifically , structural and biochemical analyses show that the second messenger c - di - gmp activates bldd dna binding by driving a unique form of dimerization that is mediated by a tetrameric form of c - di - gmp .
the c - di - gmp tetramer performs its oligomerization function by adjoining two bldd c - terminal domain ( ctd ) protomers , the polypeptide chains of which are separated by 10 .
bldd recognizes the c - di - gmp tetramer using a bipartite rxd - x8-rxxd c - di - gmp interaction signature sequence from each subunit .
thus , tetrameric c - di - gmp acts as a small - molecule dimerizing agent that controls the dna - binding activity of bldd , leading to repression of the bldd regulon of sporulation genes during vegetative growth , thereby controlling the hypha - to - spore transition in multicellular bacteria .
to gain insight into the cellular processes controlled by c - di - gmp in streptomycetes , we overexpressed either the active dgc cdgb from s. coelicolor ( tran et al . , 2011 ) or the active pde yhjh from e. coli ( pesavento et al . ,
strikingly , overexpression of both cdgb and yhjh blocked the generation of aerial mycelium by s. venezuelae ( figure 1b ) .
however , scanning electron micrographs ( sems ) revealed that , whereas overexpression of cdgb blocked development , resulting in a classical bald phenotype , overexpression of the pde yhjh in fact promoted sporulation , but the colonies appeared bald to the naked eye because aerial mycelium formation had been bypassed ( figure 1c ) .
as judged by heat resistance , the spores made by the yhjh overexpression strain were as robust as those of the wild - type ( wt ) ( figure s1a available online ) .
moreover , overexpression of catalytically inactive versions of yhjh or cdgb had no effect on s. venezuelae development ( figure s1b ) .
these data suggest that intracellular levels of c - di - gmp influence the timing of development .
in particular , they suggest that increased c - di - gmp levels delay differentiation , arresting the colonies in the vegetative growth stage , whereas decreased levels of the second messenger accelerate development , favoring sporulation .
s. venezuelae has no pilz domain - containing proteins , and no putative c - di - gmp - binding effector proteins have so far been identified in the streptomyces genus .
thus , to address the mechanism by which s. venezuelae senses c - di - gmp to control sporulation , we sought to identify c - di - gmp effector proteins involved in development . to selectively enrich putative c - di - gmp - binding proteins from s. venezuelae cell extracts
, we performed an affinity pull - down assay using a c - di - gmp capture compound ( nesper et al . , 2012 ) .
remarkably , the developmental master regulator bldd was repeatedly recovered in our c - di - gmp - based capture compound experiments .
bldd is an 18 kda dna - binding protein ( elliot and leskiw , 1999 ) consisting of two distinct domains connected by a flexible linker ( figure 2a ) .
the n - terminal domain is the dna - binding domain ( dbd ) and has a xenobiotic response element ( xre ) helix - turn - helix ( hth ) dna - binding motif ( kim et al . , 2006 ) .
the bldd ctd harbors a largely helical fold with no known function ( kim et al . , 2014 ) . to probe the interaction between bldd and c - di - gmp further and to identify the c - di - gmp - binding domain
, we used differential radial capillary action of ligand assays ( dracala ) ( roelofs et al . , 2011 ) .
dracala allows the visualization of protein - bound radiolabeled ligand as a concentrated spot after the application of the protein - ligand mixture onto nitrocellulose . using this assay
, we confirmed that full - length ( fl ) bldd ( expressed as an n - terminally his6-tagged protein ) from both s. venezuelae ( figures 2b and s2b ) and s. coelicolor ( data not shown ) bind p - labeled c - di - gmp .
importantly , the dracala assays demonstrated that the previously uncharacterized ctd of bldd functions as the c - di - gmp - binding domain ( figure 2b ) .
further , excess unlabeled c - di - gmp , but not gtp , competed with the labeled c - di - gmp for binding to fl bldd and to bldd - ctd .
thus , these data reveal that the ctd of the key developmental regulator bldd is a c - di - gmp - binding domain . using global chromatin immunoprecipitation - microarray analysis ( chip - chip )
, we previously identified the complete bldd regulon in s. coelicolor , showing that it encompasses 167 transcription units ( den hengst et al . ,
2010 ) . through meme - based sequence analysis of all the promoter regions directly targeted by bldd , we defined a 13 bp pseudo - palindromic sequence , 5-tnac(n)5gtna-3 , designated the bldd box , which functions as a specific binding sequence for bldd ( den hengst et al . , 2010 ) .
sequence analysis showed that the bldd box was conserved between s. coelicolor and s. venezuelae for most key bldd target promoters ( den hengst et al . , 2010 ) .
further , bldd from s. coelicolor and s. venezuelae contain an identical dbd and differ by only two residues in the dbd - ctd linker and five residues in the ctd , suggesting that bldd function is broadly conserved between the two species .
having shown that bldd binds c - di - gmp , we tested the effect of c - di - gmp on bldd dna binding . radiolabeled s. venezuelae dna fragments encompassing the promoter regions of two well - characterized bldd target genes , bldm and whig , including the bioinformatically identified bldd box ( figure 2c ) , were used as target dnas in electrophoretic mobility shift assays ( emsas ) ( figure 2d ) . the fixed concentration of bldd used in these assays ( 0.6 m ) was insufficient to elicit a dna mobility shift .
however , the addition of increasing concentrations of c - di - gmp ( 0.251.75 m ) strongly induced bldd binding to both the tested promoter regions ( figure 2d ) . to confirm and extend these results into cells
, we manipulated the levels of c - di - gmp in s. venezuelae and monitored the effect on bldd binding to the bldm and whig promoters in vivo using chip - sequencing ( chip - seq ) .
the degree of bldd binding was assayed at a single time point in wt s. venezuelae and the wt overexpressing either the dgc cdgb or the pde yhjh ( the strains whose phenotypes are described above ) .
consistent with the in vitro emsa data , overexpression of the dgc enhanced chip - seq peak height at the bldd target promoters relative to the wt control ( figure 2e ) .
conversely , overexpression of the pde lowered chip - seq peak heights at bldd target promoters relative to the wt ( figure 2e ) .
these data demonstrate that c - di - gmp enhances the binding of bldd to the bldd box , stimulating bldd - mediated repression of its target regulon .
thus , it is not bldd , but a bldd-(c - di - gmp ) complex , that serves to turn off sporulation genes during vegetative growth .
the opposing effects of the overexpression of the dgc cdgb and the pde yhjh suggested that high levels of c - di - gmp retard sporulation and low levels of c - di - gmp accelerate sporulation . because the bldd-(c - di - gmp ) complex serves to keep sporulation genes shut off during vegetative growth , loss of bldd
should have a similar effect on streptomyces development as depletion of c - di - gmp levels . to test this hypothesis
strikingly , the bldd null mutant formed small colonies lacking aerial hyphae , but when examined by sem even young colonies of the bldd mutant were found to contain spore chains embedded in an excess of extracellular matrix ( figure 3a ) .
heat resistance tests showed the bldd mutant spores were mildly defective ( figure s1a ) .
by contrast , equivalent young colonies of the wt that were grown and imaged in parallel had not yet developed aerial hyphae or spores ( figure 3b ) .
thus , loss of bldd mimics the effects of overexpressing the pde yhjh ( compare figures 1c and 3a ) .
in addition , overexpression of cdgb had no effect on the phenotype of the bldd mutant ( figure s2a ) , further supporting the idea that c - di - gmp signals through bldd to control the hypha - to - spore transition . to elucidate the molecular basis for c - di - gmp recognition and binding by the bldd ctd and to gain insight into how this interaction may elicit developmental signaling , we determined structures of the s. venezuelae bldd ctd ( residues 80166 ) and s. coelicolor bldd ctd ( residues 80167 ) in complex with c - di - gmp . three s. venezuelae bldd c - domain-(c - di - gmp ) structures and one s. coelicolor bldd c - domain-(c - di - gmp ) structure were determined to resolutions of 1.95 , 2.33 , 1.75 , and 2.25 , respectively ( figure 4 ) ( see extended experimental procedures and tables s1 and s2 ) .
the bldd ctd structures are composed of two ( -- ) repeats followed by a short c - terminal helix and are similar to the apo form studied by nmr ( kim et al . , 2014 ) ( figure 4 ) .
database searches revealed that the bldd ctd harbors a new fold , but reduced stringency revealed that it shows limited structural similarity with winged hth proteins , in particular , the winged hth domain of eukaryotic heat shock factor 1 ( hsf1 ) ( littlefield and nelson , 1999 ) .
the bldd ctd and hsf1 bind c - di - gmp and dna , respectively , but the motifs they employ to interact with their nucleotide ligands are completely different ( figure s3 ) .
the bldd ctd uses a previously unseen mode of c - di - gmp binding in which two noninteracting ctds are glued together by a c - di - gmp tetramer composed of two interlocked c - di - gmp dimers ( figure 4a ) .
thus , in the bldd ctd-(c - di - gmp ) complex , c - di - gmp functions as a macromolecular dimerizer .
indeed , the closest approach of any c atoms of the two tethered ctds is 10 .
the bldd ctd interacts with c - di - gmp using two contiguous surface motifs , herein called motif 1 and motif 2 , which are located between the two -- repeats .
motif 1 is composed of residues 114116 ( rgd ) and motif 2 is composed of residues 125128 ( rqdd ) ( figure 5a ) .
these motifs are located on a solvent exposed region at one end of each ctd protomer and not within a pocket or cavity ( figures 4a and 5a ) .
the combined motifs from two ctd subunits provide ideal shape and electrostatic complementarity for encasing the unusual cage - like c - di - gmp tetramer ( figures 4a and 4b ) . strikingly , although the bldd ctd is overall acidic ( pi5.0 ) , the c - di - gmp - binding surface between two bldd protomers is electropositive ( figure 4b ) .
in addition to shape and charge complementary , contacts from the arginine and aspartic acid residues within motifs 1 and 2 provide specificity for recognition of guanine cyclic nucleotides ; the multiple interactions effectively exclude binding to adenine cyclic nucleotides .
specifically , motif 2 from each ctd protomer combine to mediate contacts to one intercalated c - di - gmp dimer , asp116 of motif 1 from each protomer combine to mediate contacts to the other c - di - gmp dimer , while arg114 sits centrally and anchors both dimers ( figures 5a and 5b ) .
the guanine bases wedged between the two motif 2 regions are specified by contacts from residues arg125 and asp128 ( figures 5a and 5b ) .
residue asp128 makes two hydrogen bonds to the n1 and the exocyclic n2 atoms of the guanine bases on each end ( top and bottom layers ) of the intercalated dimer , while arg125 flanks the guanines in the center ( middle layers ) of the dimer and makes hydrogen bonds to the guanine o6 and n7 atoms ( figures 5b5d ) .
notably , the stacking interactions between the arg125 side chains are the only direct contacts between the two ctd protomers but are clearly not sufficient to promote bldd dimerization .
arg114 is the only ctd residue that makes contacts to both intercalated c - di - gmp dimers .
arg114 hydrogen bonds to the guanines contacted by asp128 , as well as the o6 atoms of the adjacent guanines of the other c - di - gmp dimer ( figures 5a and 5b ) .
thus , arg114 plays a key role in the recognition and stabilization of this unique c - di - gmp tetramer .
the c - di - gmp dimer bound between the motif 1 regions has fewer contacts and is more exposed ( figure 5a ) .
in addition to contacts from arg114 , asp116 of motif 1 hydrogen bonds to the guanine n1 and n2 atoms in a manner analogous to the contacts from asp128 of motif 2 ( figure 5 ) .
notably , the specific hydrogen bonds from motif 1 and 2 residues to guanine exocyclic o6 and n2 atoms dictate that bldd binds to c - di - gmp but not to c - di - amp , which is missing an exocyclic atom at the 2 position and harbors a hydrogen bond donor rather than an acceptor at the exocyclic 6 position .
bldd motifs 1 ( rxd ) and 2 ( rxxd ) , although similar in sequence to the inhibitory i - site ( rxxd ) , which is involved in product inhibition feedback control of dgc activity ( christen et al . ,
further , mutagenesis of either bldd motif 1 or motif 2 abolishes c - di - gmp binding in dracala assays ( figure s2b ) , confirming that , unlike i - site c - di - gmp binding , both motifs 1 and 2 in bldd are required to construct the complete binding site for the tetrameric c - di - gmp complex .
this dual signature sequence is unlike any previously characterized c - di - gmp - binding motif .
moreover , specific binding of the c - di - gmp tetramer requires encasement by two such bipartite motifs from precisely oriented bldd protomers .
while the arginine and aspartic acid residues in bldd motifs 1 and 2 dictate the c - di - gmp - binding arrangement and read the guanine bases , contacts to the c - di - gmp phosphate groups are provided by lys84 from 1 and arg130 from 3 .
further , ile110 makes van der waals interactions and residues asn118 and ser123 hydrogen bond with the guanine bases on the top and bottom layers of the c - di - gmp tetramer ( figure 5a ) .
the combination of the optimally positioned bipartite signature sequences from two bldd protomers exquisitely templates binding of the specific and unusual c - di - gmp tetramer structure .
c - di - gmp is monomeric in solution at physiological concentrations ( gentner et al . , 2012 ) .
however , intercalated c - di - gmp dimers have been observed in crystal structures of the nucleotide alone and in complexes with effector proteins .
higher order c - di - gmp structures such as tetramers and octamers have thus far only been inferred from nmr and spectroscopic studies and require very high c - di - gmp concentrations ( up to 30 mm ) and monovalent cations ( zhang et al . , 2006 ) .
these higher order structures are characterized by g - quartet interactions with a centrally bound potassium ion coordinated by four guanines .
there are minimal base contacts and no base stacking interactions in these structures ( figure s4a ) ( zhang et al . , 2006 ; gentner et al . ,
2012 ) . by sharp contrast , the bldd - bound tetrameric c - di - gmp is a tightly packed structure that is not secured by ions .
rather , the c - di - gmp molecules are closely spaced and optimally positioned for interbase pairing , leading to the formation of a multistranded , base - stacked structure with top , middle , and bottom layers ( figures 5d and s4a ) .
there are 12 hydrogen bonds between the two intercalated dimers within the c - di - gmp tetramer , including contacts between the n3 atoms and exocyclic nh2 amides of an adjacent base ( figures 5c and s4b ) .
such contacts could not be formed with c - di - amp due to its lack of an exocyclic nh2 atom .
therefore , in addition to contacts from motifs 1 and 2 , guanine - guanine base hydrogen bonds serve to specify c - di - gmp tetramer binding to bldd .
notably , formation of the c - di - gmp tetramer buries 24% of the total surface area ( buried surface area [ bsa ] ) of the c - di - gmp molecules ( figure s4b ) . by comparison , in most protein oligomers
the bsa between protomers is 15% ( wang et al . , 2009 ) .
finally , the interface between the intercalated c - di - gmp dimers that forms the tetramer is remarkably complementary in shape ( figure s4b ) .
thus , the combination of multiple contacts between the c - di - gmp moieties along with its extensive bsa and molecular shape complementarity lead to the creation of a compact and highly specific c - di - gmp tetramer . however , bldd is necessary to stabilize this tetramer and template its formation .
the bldd ctd-(c - di - gmp ) crystal structures reveal that c - di - gmp acts as a dimerizer to link two ctd protomers . to examine the effect of c - di - gmp on the oligomeric state of the bldd ctd in solution , we carried out chemical crosslinking and size exclusion chromatography ( sec ) studies .
chemical crosslinking experiments were performed using disuccinimidyl suberate ( dss ) , which contains amine - reactive n - hydroxysuccinimide esters at both ends of an 11 spacer arm .
this reagent should therefore be able to crosslink even the distantly anchored ctd protomers observed in our ctd-(c - di - gmp ) structures ( figure 4a ) . in the absence of dss , the bldd dbd and ctd migrate on sds - page gels as single bands with the expected monomeric molecular weights of 11 and 12 kda , respectively ( figure 6a ) .
upon incubation with dss , the bldd dbd forms a covalent dimer of 21 kda , consistent with previous biochemical and structural analyses of this domain ( kim et al . , 2006 ; lee et al . , 2007a ) , and oligomerization is unaffected by the addition of c - di - gmp ( figure 6a ) .
however , in the presence of c - di - gmp , addition of dss results in the clear formation of covalent ctd dimers ( figure 6a ) . to examine c - di - gmp - induced ctd oligomerization further , we performed sec analyses .
as part of this study , we mutagenized the rgd ( motif 1)-x8-rqdd ( motif 2 ) c - di - gmp - binding signature of the bldd ctd . to retain the charge of these surface residues
, we made the charge - swapped dgr - x8-dqdr ctd mutant , changing the key arg residues within the two motifs to asp , and vice versa .
dracala assays showed that this mutant ctd was unable to bind c - di - gmp ( figure s2b ) .
sec experiments in the presence of 3 m c - di - gmp showed that the wt ctd forms a dimer , while the mutant ctd is monomeric ( figure 6b ) .
hence , the crosslinking and sec data support our structural finding that c - di - gmp is required for bldd ctd dimerization .
further , we constructed a bldd mutant allele encoding a protein solely defective in c - di - gmp binding ( carrying the dgr - x8-dqdr mutation ) and found that it had no ability to complement a bldd mutant ( figure s2a ) , confirming that the major in vivo functions of bldd are indeed mediated by its binding to c - di - gmp .
our structures reveal a unique interaction between the bldd ctd and a c - di - gmp tetramer .
remarkably , every guanine in this complex is read specifically by either bldd arginines , aspartic acids , and/or other guanine bases , suggesting that bldd binds only c - di - gmp , and not other cyclic nucleotides . to test this hypothesis and further probe the c - di - gmp - binding affinity of bldd , we performed fluorescence polarization ( fp ) experiments .
these studies were performed with bldd ctd that was expressed and purified from sf9 insect cells to ensure no c - di - gmp was present , as eukaryotes do not produce c - di - gmp ( extended experimental procedures ) .
the bldd-(c - di - gmp ) structures show that one ribose of each c - di - gmp bound to bldd must be unmodified to permit formation of the bldd-(c - di - gmp ) complex ( figure s5a ) .
hence , for these studies we used the fluoresceinated probe , 2-fluo - ahc - c - di - gmp , which harbors the fluorescein dye on only one ribose ( figure s5a ) .
these studies revealed bldd ctd bound to 2-fluo - ahc - c - di - gmp , with an apparent kd of 2.5 m 0.6 ( figure s5b ) .
consistent with our dracala assays ( figure s2b ) , the dgr - x8-dqdr ctd mutant failed to bind 2-fluo - ahc - c - di - gmp in fp assays ( figure s5b ) .
wt bldd ctd showed no binding to the identically fluoresceinated c - di - amp tagged molecule , 2-fluo - ahc - c - di - amp ( figure s5b ) .
next , to ascertain the stoichiometry of c - di - gmp binding in solution , we used an fp - based binding assay . the resulting data ( figure s5c )
a single inflection point can be fitted at a bldd monomer concentration of 12 m , which equates to a stoichiometry of four c - di - gmp molecules per ctd dimer . however
, careful inspection of the data reveals another potential inflection point at a bldd monomer concentration of 6 m , which would be consistent with an initial binding event of two c - di - gmp molecules per ctd dimer .
this putative initial binding event is also apparent in the equilibrium - binding isotherm yielding an apparent kd of 1.7 m ( figure s5b ) .
two - step binding is not inconsistent with the structure , but the nearly identical affinities observed for each binding event suggest positive cooperativity .
overall , these data demonstrate unequivocally that c - di - gmp binds the ctd with a stoichiometry of four c - di - gmp molecules per ctd dimer , concordant with our structures .
these studies also demonstrate that the bldd ctd binds specifically and with high affinity to c - di - gmp but not c - di - amp and that both motifs 1 and 2 are essential for this interaction .
dimeric bldd binds pseudo - palindromic dna sites that contain a 5-tnac(n)5gtna-3 consensus ( den hengst et al . ,
2010 ) . our data and those of others ( lee et al . ,
2007a ) show that the bldd dbd alone can dimerize at higher concentrations ( 10 m ) .
consistent with these findings , the crystal structure of the s. coelicolor bldd dbd revealed a dimer with a small contact interface ( kim et al . , 2006 ) .
we obtained additional views of the bldd dbd by solving the s. venezuelae bldd dbd structure to 2.80 resolution .
comparison of these dimers with the dimer from the s. coelicolor dbd structure showed that , although hydrophobic residues within the c - terminal regions of the dbds make contacts between the subunits in each case , all three dimers take distinct conformations ( figure s6a ) .
moreover , there is less than 300 bsa per subunit in this dimer , which is far less than the 1000 bsa per subunit typically observed for biologically relevant dimers ( krissinel and henrick , 2007 ) .
these data indicate that the bldd dbd is unlikely to form a stable dna - binding active dimer at physiologically relevant concentrations .
in addition , although the bldd dbd resembles the equivalent dbd of repressor , previous modeling studies suggested that the bldd dbds would not interact specifically with dna if bldd employed a dna - binding mechanism similar to that utilized by repressor ( kim et al . , 2006 ) .
our finding that c - di - gmp binding leads to the formation of a c - di - gmp bridged ctd dimer provides the missing link to this puzzle .
however , how c - di - gmp binding to the ctds is signaled to the dbds to bring about dna binding remained unclear . to deduce the mechanism by which c - di - gmp activates bldd to bind dna , we determined the structure of a bldd-(c - di - gmp)-21-mer dna complex to 4.5 resolution ( extended experimental procedures ; figure s6b ) . while the low resolution of the structure precludes a detailed analysis , the electron density maps show the overall arrangement of the domains and how the dbds dock onto the dna .
critically , the structure reveals that bldd binding to cognate dna is more similar to the dna binding mode of the xre protein sinr ( lewis et al . , 1998 ;
2013 ) than to that of the repressor , as the two bldd dbds are juxtaposed when bldd is bound to dna ( figures 7a and 7b ) .
the dbd - dbd interacting surfaces observed in the bldd - dna complex correspond to the hydrophobic regions near the dbd c terminus that interact in the apo dbd structures .
however , the interfaces are yet again different , supporting the notion that dbd dimerization is weak and malleable .
such malleability is critical to allow the dimeric bldd hth elements to bind the dna , which is bent by 30. the bldd dbds are tethered to the ctd via a linker that was previously shown to be highly flexible ( kim et al .
not surprisingly , this linker ( pgttpggaaeppp ; residues 7184 ) is disordered in the bldd-(c - di - gmp)-21-mer structure .
its flexibility is underscored by the different orientation of the two ctds in the structure relative to the dbd - dna complex ( figure 7a ) .
the ctd subunits in the dimer make different interactions that help anchor them in the bldd-(c - di - gmp)-dna crystal ; one of the ctds interacts with a hydrophobic patch on its cognate dbd , while the other ctd makes crystal contacts with a symmetry mate ( figures 7a and s6c ) .
however , the ctds in the fl bldd-(c - di - gmp)-dna structure are dimerized in a manner identical to that observed in our ctd-(c - di - gmp ) structures ( figures 4d , 5a , 7a , and s6c ) .
additional evidence that the linker region between the dbd and ctd is flexible was provided by proteolysis experiments employing endoproteinase glu - c , which cleaves exposed peptide bonds on the carboxyl side of glutamic or aspartic acid residues .
hence , if the bldd linker region is unstructured , glu - c should selectively cleave after bldd residue glu80 .
glu - c proteolysis experiments were carried out on the fl bldd protein , the fl protein with the 21-mer dna present , and the fl protein in the presence of both c - di - gmp and the 21-mer dna . in all three cases bldd
was readily cleaved into two bands , corresponding to the dbd and the ctd ( figure s6d ) . thus , neither the presence of dna , nor of c - di - gmp and dna , protected bldd from proteolysis , indicating the linker is exposed even in the presence of cognate ligands . therefore ,
the combined data suggest a molecular model for c - di - gmp activation of bldd dna binding in which binding of c - di - gmp leads to the formation of a c - di - gmp - linked bldd ctd dimer ( figure 7c ) .
such ctd dimerization effectively brings the two dbds into proximity , thereby increasing their local concentration to allow germane dbd dimerization on cognate dna ( figure 7c ) .
the inherent flexibility afforded by the dbd - ctd linker allows the dbds to adjust for optimal binding to multiple pseudo - palindromic bldd dna boxes .
the role of c - di - gmp has been studied extensively in unicellular gram - negative bacteria , in which most c - di - gmp - dependent signaling pathways control the transition from a planktonic , motile lifestyle to a surface - associated , sessile lifestyle ( stick or swim ) . here
we show that the activity of the streptomyces master regulator bldd is also controlled by c - di - gmp , thus bringing the regulatory role of this key second messenger into a new physiological arena , that of differentiation in gram - positive multicellular bacteria .
our studies indicate that c - di - gmp binding to bldd controls the developmental switch between vegetative growth and sporulation .
specifically , we demonstrate that c - di - gmp binding to bldd activates its dna - binding activity , which results in repression of sporulation genes during vegetative growth .
consistent with this , genetic studies revealed that bldd null mutants sporulate precociously , mimicking the effect of overexpressing a c - di - gmp phosphodiesterase .
thus , c - di - gmp signals through bldd to control the hypha - to - spore developmental transition in streptomyces .
few c - di - gmp effector - binding motifs have been identified to date .
2009 ; lee et al . , 2007b ; petters et al . , 2012 ) , inactive eal domains ( navarro et al .
, 2009 ; qi et al . , 2011 ; newell et al . ,
structures have shown that c - di - gmp interacts with these motifs as either a monomer or intercalated dimer .
the bldd protein does not contain any previously characterized c - di - gmp effector - binding motifs .
thus , to elucidate the mechanism by which c - di - gmp acts as a switch to turn on the dna - binding activity of bldd , we determined several structures of the bldd ctd complexed to c - di - gmp as well as a 4.5 structure of the bldd-(c - di - gmp)-dna complex .
these structures revealed that bldd interacts with c - di - gmp using a heretofore unseen c - di - gmp binding mode involving a unique c - di - gmp - binding signature sequence consisting of two proximal arginine and aspartic acid containing motifs , motif 1 ( rxd ) and motif 2 ( rxxd ) , separated by eight residues .
remarkably , in this binding mode , a tetrameric form of the c - di - gmp functions as a small - molecule dimerizer to adjoin two noninteracting bldd protomers .
notably , the identical ctd dimer-(c - di - gmp ) tetramer structure was seen in multiple crystal forms .
finally , binding studies confirmed that the bldd ctd binds c - di - gmp with a stoichiometry of four c - di - gmp molecules to one bldd ctd dimer .
the c - di - gmp tetramer revealed in these structures represents a previously unknown form of this nucleotide second messenger .
indeed , we do not know of any other example in which a signaling molecule can assume different oligomeric states to effect its function .
bldd is present throughout the sporulating actinomycetes ( den hengst et al . , 2010 ) , including , for example , nitrogen - fixing frankia that live in symbiosis within the root nodules of alder trees , and members of the marine genus salinospora , which have recently emerged as an important source of antibiotics and other medically significant compounds . outside of the genus streptomyces ,
the only actinomycete in which bldd has been investigated is saccharopolyspora erythraea , where bldd directly controls expression of the biosynthetic cluster of the clinically important antibiotic erythromycin ( chng et al . , 2008 ) .
homologs from across the sporulating actinomycetes share 77%99% sequence identity with s. venezuelae bldd .
the main region of conservation between these proteins is the n - terminal dna - binding domain , which shares 95%100% identity .
although our bldd-(c - di - gmp)-dna structure is too low resolution to ascribe specific protein - dna contacts , it reveals the location of the hth motif and residues that likely contact the dna .
notably , these amino acids are the most conserved among bldd homologs ( essentially 100% ; figure s7 ) . by contrast , the ctd regions of bldd proteins are less well conserved ( as low as 48% identity ) .
hence , it is striking , given this low conservation , that the residues that interact with c - di - gmp are strictly conserved ( figure s7 ) .
the only exception is lys84 , which contacts c - di - gmp phosphate groups .
however , in all bldd homologs this residue is either a lysine or arginine and thus able to make the same electrostatic interaction . of particular note , residues r114 , d116 , r125 , and d128 ( from motifs 1 and 2 ) , which mediate essential specifying contacts with c - di - gmp ,
further , all of the actinomycetes that encode an ortholog of bldd also encode ggdef domain - containing dgcs .
these combined findings indicate that bldd-(c - di - gmp ) is likely to control key developmental processes throughout the sporulating actinomycetes , using tetrameric c - di - gmp as a second messenger .
for a full explanation of the experimental protocols , see extended experimental procedures in supplemental information .
strains and plasmids used are shown in table s3 , and oligonucleotides used are shown in table s4 .
a bldd null mutant ( sv77 ) was constructed by redirect pcr targeting , and the bldd::apr mutant allele was moved into a new wt background by generalized transduction using the s. venezuelae - specific phage sv1 .
transduction of the bldd::apr allele was confirmed by pcr and the strain was named sv74 .
cyclic di - gmp capture compound experiments were performed as described previously ( nesper et al . , 2012 ) but with the minor modifications described in extended experimental procedures .
briefly , the c - di - gmp capture compound was added to the soluble lysates and , following uv irradiation in the caprobox , magnetic streptavidin beads were added to the reaction .
after incubation the beads were collected , washed , and boiled in sample buffer , the proteins released were run on sds - pa gels and cut out for mass spectrometry analysis .
the dracala assays used his6-bldd or n - terminally his - tagged domains , which were incubated with 11 nm p - c - di - gmp .
the competition experiments had 266 m cold c - di - gmp or gtp added to the reaction .
dna fragments spanning the bldm ( 158 bp ) and whig ( 151 bp ) promoter regions of s. venezuelae were generated by pcr and 5 end labeled using [ -p]-atp and t4 polynucleotide kinase .
the binding reactions were performed using 0.6 m his6-bldd and radiolabeled dna ( 8,000 cpm ) as well as 0.5 g poly[d(i - c ) ] as nonspecific competitor dna .
when appropriate , increasing amounts of c - di - gmp ( 0.251.75 m ) were added to the mixture .
the reaction samples were incubated for 20 min at room temperature and then run on 5% polyacrylamide gels .
chromatin immunoprecipitations were performed as described ( bush et al . , 2013 ) using an anti - bldd polyclonal antibody .
the oligomeric states of bldd and its domains were analyzed via chemical crosslinking using dss in the presence and absence of c - di - gmp ( see the extended experimental procedures ) and visualized on sds - pa gels .
molecular weight analyses using sec experiments were performed with a hiload 16/600 superdex 75 pg column . for detailed descriptions of the protein expression , purification , crystallization , structure determination , and refinement protocols ,
see the extended experimental procedures . extended experimental proceduresbacterial strains , growth conditions , and conjugationsall e. coli strains used in this study ( table s3 ) were grown in lb medium under aeration at 37c .
e. coli dh5 was used for plasmid and cosmid propagation and bl21(de3)plyss for protein overexpression .
bw25113 ( datsenko and wanner , 2000 ) containing a red plasmid , pij790 , was used to create the bldd disruption cosmid and et12567 containing puz8002 ( paget et al . , 1999 )
s. venezuelae strains ( table s3 ) were grown at 30c on maltose - yeast extract - malt extract ( mym ) medium ( stuttard , 1982 ) containing 50% tap water ( mym - tap ) and 200 l trace element solution ( kieser et al . , 2000 ) per 100 ml .
conjugations between e. coli and s. venezuelae were carried out as previously described ( bibb et al .
, 2012).construction of plasmidsthe oligonucleotides used for plasmid constructions are listed in table s4 . for protein overexpression and purification , bldd and its individual domains
were cloned into pet15b resulting in n - terminally his - tagged fl ( full - length ) bldd ( amino acid residues 1 - 166 ) , bldd - dbd ( amino acid residues 1 - 79 ) and bldd - ctd ( amino acid residues 80 - 166 ) .
for overexpression of yhjh in s. venezuelae , an n - terminally codon optimized variant of yhjh was cloned downstream of the ermep promoter in the bt1 attb site - specific integrative vector pij10257 ( hong et al . , 2005 ) .
point mutations in bldd were introduced by following the four - primer / two - step pcr protocol ( germer et al . , 2001 ) .
for complementation analysis the bldd gene carrying its native promoter and the r114d , d116r , r125d and d128r mutations was expressed from the integrative vector pms82.construction of a bldd null mutant derivative of s. venezuelae and phage transductionthe bldd mutant was generated according to the redirect pcr targeting protocol ( gust et al . , 2003 , 2004 ) .
the sv-4-h05 cosmid was introduced into e. coli bw25113 and bldd was replaced with the apramycin - resistance ( apr ) cassette containing orit , which was amplified from pij773 using primers with bldd - specific extensions ( table s4 ) .
the disrupted cosmid was confirmed by restriction and pcr analyses and introduced into e. coli et12567/puz8002 for conjugation into s. venezuelae .
a null mutant generated by double crossing over was identified by its apramycin - resistant and kanamycin - sensitive phenotype and named sv77 after confirmation by pcr using test primers listed in table s4 .
the mutant allele bldd::apr was moved into a new wt background by generalized transduction using the s. venezuelae - specific phage sv1 ( stuttard , 1979 ) . to prepare sv1-lysate ,
10 phage were added to 10 sv77 donor spores in 800 l pre - warmed ( 45c ) soft nutrient agar ( sna ) and poured onto difco nutrient agar plates containing 0.5% glucose , 10 mm mgso4 and 10 mm ca(no3)2 .
the plates were incubated at 30c overnight , then flooded with 2.5 ml difco nutrient broth ( dnb ) and incubated for 3 - 4
the phage - containing dnb soak - out was harvested and filtered through a 0.45 m filter to eliminate bacterial contamination .
for transduction of the bldd::apr allele , 10 phage particles harvested from the bldd::apr mutant strain sv77 , were mixed with 10 - 10 wt spores and incubated overnight on mym agar at room temperature before overlaying with apramycin for selection .
transduction of the bldd::apr allele was confirmed by pcr using test primers listed in table s4 , and the strain was named sv74.c - di - gmp protein capture experimentscyclic di - gmp capture compound experiments were performed as described previously ( nesper et al . , 2012 ) with minor modifications .
s. venezuelae cultures were grown in mym - tap supplemented with trace element solution until late transition phase for 24 hr at 30c and then pelleted by centrifugation for 10 min at 6,000 rpm .
the pellet was resuspended in lysis buffer ( 6.7 mm mes , 6.7 mm hepes , 200 mm nacl , 6.7 mm potassium acetate ( kac ) , ph 7.5 ) containing protease inhibitor and dnase i. cells were put through a french press four times at 18,000 psi and then centrifuged at 100,000 x g for 1 hr . for the capture experiments , the protein concentration of the soluble fraction
was determined using a uv / vis nanodrop spectrophotometer and 400 g protein were mixed with 10 m c - di - gmp capture compound and with 20 l 5 x capture buffer ( 100 mm hepes , 250 mm kac , 50 mm magnesium acetate ( mgac ) , 50% glycerol , ph 7.5 ) .
1 mm c - di - gmp was added to the control reaction and incubated for 30 min prior to capture compound addition .
the reaction volume was adjusted with h2o to 100 l and incubated for 2 hr at 4c in the dark on a rotary wheel .
after uv irradiation for 4 min in a caprobox , 50 l magnetic streptavidin beads and 25 l 5 x wash buffer ( 250 mm tris ph 7.5 , 5 m nacl , 0.1% n - octyl--glucopyranoside ) were added to the reaction and the mixture was incubated for 45 min at 4c on a rotary wheel .
the beads were then collected with a magnet and washed 6 times with 200 l wash buffer .
the beads were resuspended in 20 l sample buffer and run for 10 min on a 15% sds polyacrylamide gel after 10 min incubation at 95c .
the gel was stained using instantblue coomassie stain solution , and a 11 cm gel slice containing all captured proteins was excised for analysis by mass spectrometry.electrophoretic mobility shift assaysdna fragments spanning the bldm ( 158 bp ) and whig ( 151 bp ) promoter regions of s. venezuelae were generated by pcr using oligonucleotides listed in table s4 and then 5 end - labeled using [ -p]-atp and t4 polynucleotide kinase .
the binding reactions were performed in bandshift buffer ( 10 mm tris ph 7.5 , 1 mm edta , 5% glycerol , 10 mm nacl , 1 mm mgcl2 ) in 20 l reaction mixture containing 0.6 m bldd and radiolabeled dna ( 8,000 c.p.m . ) as well as 0.5 g poly[d(i - c ) ] as nonspecific competitor dna .
when appropriate , increasing amounts of c - di - gmp ( 0.25 1.75 m ) were added to the mixture .
the reaction samples were incubated for 20 min at room temperature followed by electrophoresis on a 5% polyacrylamide gel in 0.5 x tbe ( tris - borate - edta ) buffer at 80v for 105 min .
the gels were dried before being analyzed on a phosphorimager.bldd chip - seq experimentss .
chromatin immunoprecipitations were performed as previously described ( bush et al . , 2013 ) , except that an anti - bldd polyclonal antibody was used and pulled down with protein a - sepharose beads . library construction ,
sequencing and chip - seq data analyses were all carried out as previously described ( bush et al .
, 2013).chemical crosslinking and sds polyacrylamide gel electrophoresisthe his6-bldd - dbd and his6-bldd - ctd proteins were dialyzed into crosslinking buffer ( 100 mm nah2po4 , 150 mm nacl , ph 8) and then incubated at room temperature for 30 min in 20 l reaction samples containing 10 m protein , 1 mm disuccinimidyl suberate ( dss ) in dimethylsulfoxide ( dmso ) , and c - di - gmp as indicated .
the reaction was stopped by adding 50 mm tris ph 8 and incubation for 15 min followed by addition of sds sample buffer and heating to 95c for 10 min .
samples were separated on a 15% sds polyacrylamide gel and visualized by coomassie staining.determination of c - di - gmp binding to proteins by differential radial capillary action of ligand assayradiolabeled c - di - gmp was synthesized in vitro using [ -p]-gtp and the purified diguanylate cyclase pled as described ( paul et al . , 2004 ) .
the dracala assays ( roelofs et al . , 2011 ) were performed using 2 g of his6-bldd or its n - terminally his - tagged domains that were incubated with 11 nm p - c - di - gmp in dgc buffer ( 250 mm nacl , 25 mm tris ph 8 , 10 mm mgcl2 , 5 mm -mercaptoethanol ) . for competition experiments ,
266 m cold c - di - gmp or gtp were added to the reaction . after a 5 min incubation at room temperature ,
5 l of the binding sample were spotted onto nitrocellulose membrane and the dried membranes were analyzed using a phosphorimager.purification , crystallization , and structure determination of s. venezuelae and s. coelicolor bldd ctd-(c - di - gmp ) complexesfor structural studies on the ctd , the regions encoding residues 80 - 166 ( s. venezuelae bldd ) and 80 - 167 ( s. coelicolor bldd ) were cloned into the pet15b vector and the proteins induced at 37c and purified via ni - nta chromatography .
the his - tags were removed from the proteins used for structural studies by thrombin cleavage .
crystals of the s. venezuelae bldd ctd-(c - di - gmp ) complex , which assumed the trigonal space group , p32 , were obtained using protein at 30 mg / ml and 1 mm c - di - gmp .
crystals were produced via the hanging drop vapor diffusion method and mixing the complex 1:1 with 28% peg 1500 , 100 mm sodium acetate , ph 5.5 .
the s. venezuelae bldd ctd contains no methionines and hence for phasing , leu92 and ile135 were substituted with methionines .
semet(l92m / i135 m ) bldd ctd was expressed using the methionine inhibitory pathway and the protein purified and crystallized with c - di - gmp as per the wt ctd .
the selenomethionine - substituted l92m / i135 m protein crystallized in the wild - type protein p32 space group .
a second crystal form of the s. venezuelae bldd ctd-(c - di - gmp ) complex was grown with protein at 20 - 40 mg / ml and 1 mm c - di - gmp using 20% peg 2000 monomethyl ether , 100 mm mes , ph 6.0 , as a crystallization reagent and took the orthorhombic space group p21212 .
the third crystal form of the s. venezuelae bldd ctd-(c - di - gmp ) complex was produced with protein at 10 mg / ml and 1 mm c - di - gmp using 1.2 m sodium / potassium phosphate , 50 mm citrate ph 5.6 .
crystals were obtained for the s. coelicolor bldd ctd-(c - di - gmp ) complex using 25 mg / ml protein , 1 mm c - di - gmp and mixing the complex 1:1 with a reservoir comprised of 1.4 m sodium / potassium phosphate , 100 mm hepes ph 7.5 .
these crystals take the c2221 space group.multiple wavelength anomalous diffraction ( mad ) data were collected for a semet(l92m / i135 m ) s. venezuelae bldd ctd-(c - di - gmp ) crystal to 2.28 resolution at als ( advanced light source , berkeley , ca , usa ) beamline 8.3.1 ( table s1 ) .
the data were processed using mosflm and the heavy atom substructure was obtained via solve ( terwilliger and berendzen , 1999 ) .
phenix was used for final phasing and density modification ( adams et al . , 2010 ) .
the crystal contains 12 protein molecules in the asymmetric unit ( asu ) and each of the six dimers is glued together by four c - di - gmp molecules .
the six c - di - gmp complexed dimers are essentially identical ( figure 4d ) .
final refinement was done using a data set collected to 1.95 resolution for a semet(blddl92 m ) ctd-(c - di - gmp ) crystal .
a wt data set was also collected to 2.2 resolution and the structure was identical to the l92 m and l92m / i135 m structures .
the final 1.95 resolution - structure contains residues 84 - 161 for each of the 12 subunits and 24 c - di - gmp molecules ( table s2 ) .
data were collected to 1.75 , 2.25 and 2.33 resolution for the s. venezuelae and s. coelicolor bldd ctd-(c - di - gmp ) c2221 forms and the s. venezuelae bldd ctd-(c - di - gmp ) p21212 crystal form , respectively , and the structures solved by molecular replacement ( mr ) .
the s. coelicolor bldd ctd and s. venezuelae bldd ctd c2221 crystal forms contain a ctd dimer and four c - di - gmp molecules in the asu and the s. venezuelae bldd c - domain-(c - di - gmp ) p21212 crystal form contains 10 subunits ( five dimers ) , and 20 c - di - gmp molecules .
the structures were solved by mr using the program phaser ( mccoy et al . , 2007 ) .
the topology of every bldd ctd is 1 ( residues 84 - 88)-1 ( residues 89 - 93)-2 ( residues 98 - 113)-2 ( residues 120 - 124)-3 ( residues 128 - 136)-4 ( residues 140 - 149)-5 ( residues 154 - 160).crystallization and structure determination of the s. venezuelae bldd dbdthe bldd dna binding domain ( bldd dbd ) , encoding residues 1 - 79 , was cloned into pet15b , expressed in e. coli bl21(de3 ) and the protein purified via ni - nta chromatography .
prior to crystallization , the hexa - his tag was removed via thrombin cleavage and the protein further purified by size exclusion chromatography .
crystals were grown by mixing the protein ( 40 mg / ml ) 1:1 with a reservoir consisting of 35% peg 400 , 0.1 m mgcl2 and 0.1 m tris ph 7.5 .
x - ray intensity data were collected to 2.8 resolution at als beamline 8.3.1 and processed with mosflm .
the rsym and i/(i ) for the data are 11.3% ( 38.4% ) and 11.4 ( 4.3 ) , respectively , where the values in parentheses indicate data from the highest resolution shell .
the structure , which contains 3 subunits in the asu ( 2 subunits form a dimer and crystal symmetry generates a second dimer ) , was solved with phaser using a single s. coelicolor dbd subunit ( pdb code 2ewt ) as the search model .
the final model contains residues 3 - 71 of each subunit and was refined using phenix to final rwork / rfree values of 23.7%/28.9% , respectively ( adams et al .
, 2010).crystallization and structure determination of the s. venezuelae bldd-(c - di - gmp)-21-mer complexcrystals of the full - length ( fl ) s. venezuelae bldd-(c - di - gmp)-21-mer complex were grown by using protein in which the n - terminal hexa - his tag had been cleaved and incubated with 1 mm c - di - gmp ( final concentration ) .
this bldd-(c - di - gmp ) solution was mixed in a 1:1 molar ratio of bldd dimer to 21-mer dna duplex ( 5-cccctcacgctgcgtgacggg-3 , with the canonical bldd box underlined , annealed to its complementary oligodeoxynucleotide ) for crystallization trials .
crystals were grown by mixing the protein - dna complex 1:1 with the crystallization solution composed of 100 mm sodium citrate tribasic / citric acid ph 4.0 and 200 mm ammonium sulfate .
the crystals take the trigonal space group , p3221 , with a = b = 114.0 , c = 95.2 and contain a bldd dimer-(c - di - gmp)-21-mer duplex in the asu .
data were collected to the limiting resolution of 4.5 . the rsym = 9.0% ( 86.0% ) and i/(i ) = 5.8 ( 1.8 ) , where the values in parentheses are for the highest resolution shell .
first , bldd dna binding domain - dna complex models were constructed based on the sinr - dna ( pdb code 3zkc ) or repressor - dna ( pdb code 1lmb ) complex structures and used in the mr program phaser ( mccoy et al . , 2007 ) .
this solution was then used as a static model with the ctd-(c - di - gmp ) dimeric structure as a search model .
due to its low resolution , the structure was subjected to rigid body refinement only ( rwork / rfree = 33.0%/38.9% , respectively).proteolysis of fl bldd by endoproteinase glu - cto examine the flexibility of the linker region that connects the bldd dbd and ctd , limited proteolysis experiments were carried out .
specifically , the accessibility of residue glu80 , which is the only acidic residue in the bldd linker region , was determined by the ability of the glu - c protease to cleave after this residue . in these experiments , endo - glu - c ( 100 units / ml ) was a diluted 50 fold into samples of 1 ) fl bldd ( 1 mg / ml ) , 2 ) fl bldd-21-mer dna ( 1 mg / ml protein with 100 m dna ) or 3 ) fl bldd-(c - di - gmp)-21-mer ( 1 mg / ml protein with 100 m 21-mer dna and 1 mm c - di - gmp ) .
the proteolysis buffer was 50 mm sodium phosphate , ph 7.5 and the final protein concentration of each sample was 4 mg / ml .
the samples shown in figure s6d were taken at a time point of 4 hr .
notably , proteolysis of each sample was identical , demonstrating that ligand binding of neither cognate dna nor c - di - gmp by bldd affect the accessibility of the linker region , which remains flexible and unstructured.size exclusion chromatographic analyses of wild - type bldd ctd and the bldd ctd quadruple mutantsize exclusion chromatography experiments were carried out using a hiload 16/600 superdex 75 pg column .
5 mg of either the wild - type bldd ctd or the quadruple mutant were loaded onto a column that had been pre - equilibrated with 150 mm nacl , 5% glycerol , 20 mm tris hcl ph 7.5 and 3 m c - di - gmp .
the samples were run and eluted with the same c - di - gmp containing buffer .
the elution volume was plotted against a standard curve to determine the relative molecular weights of the samples .
the standard curve was determined using cytochrome c ( 12 kda ) , carbonic anhydrase ( 29 kda ) and albumin ( 66 kda).cryo - scanning electron microscopycryo - sem was performed as previously described ( bush et al . , 2013).bldd ctd expression and purification from sf9 cellswhen induced in e. coli bl21(de3 ) at 37c , bldd ctd samples contained little to no c - di - gmp contamination , which was supported by a280/a260 values of the purified protein , which were approximately 1.6 .
however , to ensure that there was no endogenous c - di - gmp present in samples used for binding affinity measurements , the bldd ctd was expressed and purified in sf9 insect cells . for these studies , a gene encoding the same s. venezuelae bldd ctd region that was expressed in e. coli was codon optimized for expression in insect cells ( genscript , piscataway , nj , usa ; http://www.genscript.com ) and subcloned into the expression vector f1 , which was transfected into the dh10bac strain for generation of the recombinant bacmid .
rbacmids were then transfected in sf9 insect cells with cellfectin ii and the cells incubated in sf-900 ii sfm for 56 days before harvest .
the supernatant was collected for the p1 viral stock and p2 was amplified for later infection .
sf9 cells expressing bldd ctd were harvested at 72 hr post infection and cells were lysed into 25 mm tris ph 7.5 , 300 mm nacl and 5% glycerol with protease inhibitors .
the protein was purified from the supernatant by ni - nta followed by size exclusion chromatography and was > 90% pure as assessed by sds - page analysis.determination of the affinity , stoichiometry , and specificity of c - di - gmp for sf9-purified bldd ctd by fluorescence polarizationto measure binding , 2-o-(6-[fluoresceinyl]aminohexylcarbamoyl)-cyclic diguanosine monophosphate ( 2-fluo - ahc - c - di - gmp ) , was used as the fluoresceinated ligand .
this molecule is conjugated via a 9 atom spacer to one of the 2 hydroxyl groups of the c - di - gmp , hence meeting the structural requirement for bldd binding that only one 2 hydroxyl group be unmodified and the other available for the interactions observed in the bldd-(c - di - gmp ) structures ( figure 5 ) .
binding was carried out at 25c in a buffer of 150 mm nacl and 25 mm tris - hcl ph 7.5 , which contained 1 nm 2-fluo - ahc - c - di - gmp .
increasing concentrations of bldd ctd were titrated into the reaction mixture to obtain the binding isotherms .
after each addition of protein the reaction sample was incubated for 30 min to ensure equilibrium had been reached .
c - di - gmp binding to bldd appears to have the characteristics of a high affinity / slow binding ligand .
the resulting data were plotted using kaleidagraph and curves were fitted to deduce binding affinities . to determine the binding stoichiometry of the bldd ctd-(c - di - gmp ) complex , the same fp binding conditions were used but the total concentration of c - di - gmp ( c - di - gmp + 2-fluo - ahc - c - di - gmp ) was 25 m , 10-fold above the kd of 2.5 m ensuring stoichiometric binding .
close inspection of the binding isotherm revealed the possibility that there are two nearly identical binding events ( likely corresponding to the binding of each intercalated c - di - gmp dimer ) .
the graph of the resulting data shows a linear increase in the observed mps until saturation of the binding sites , after which the line is flat .
importantly , the final inflection occurs at a bldd monomer concentrations of 12 m , which , when divided by the concentrations of c - di - gmp ( 25 m ) , indicates a stoichiometry of two ctd protomers per four c - di - gmps .
as anticipated from a purely chemical complementarity - orientated argument , fp studies carried out in the same buffer also revealed that wild - type bldd ctd does not bind to c - di - amp .
in addition , the bldd dgr - x8-dqdr ctd mutant did not bind the c - di - gmp probe .
all e. coli strains used in this study ( table s3 ) were grown in lb medium under aeration at 37c .
e. coli dh5 was used for plasmid and cosmid propagation and bl21(de3)plyss for protein overexpression .
bw25113 ( datsenko and wanner , 2000 ) containing a red plasmid , pij790 , was used to create the bldd disruption cosmid and et12567 containing puz8002 ( paget et al . , 1999 )
s. venezuelae strains ( table s3 ) were grown at 30c on maltose - yeast extract - malt extract ( mym ) medium ( stuttard , 1982 ) containing 50% tap water ( mym - tap ) and 200 l trace element solution ( kieser et al . , 2000 ) per 100 ml .
conjugations between e. coli and s. venezuelae were carried out as previously described ( bibb et al . , 2012 ) .
for protein overexpression and purification , bldd and its individual domains were cloned into pet15b resulting in n - terminally his - tagged fl ( full - length ) bldd ( amino acid residues 1 - 166 ) , bldd - dbd ( amino acid residues 1 - 79 ) and bldd - ctd ( amino acid residues 80 - 166 ) .
for overexpression of yhjh in s. venezuelae , an n - terminally codon optimized variant of yhjh was cloned downstream of the ermep promoter in the bt1 attb site - specific integrative vector pij10257 ( hong et al . , 2005 ) .
point mutations in bldd were introduced by following the four - primer / two - step pcr protocol ( germer et al . , 2001 ) .
for complementation analysis the bldd gene carrying its native promoter and the r114d , d116r , r125d and d128r mutations was expressed from the integrative vector pms82 .
the bldd mutant was generated according to the redirect pcr targeting protocol ( gust et al .
the sv-4-h05 cosmid was introduced into e. coli bw25113 and bldd was replaced with the apramycin - resistance ( apr ) cassette containing orit , which was amplified from pij773 using primers with bldd - specific extensions ( table s4 ) .
the disrupted cosmid was confirmed by restriction and pcr analyses and introduced into e. coli et12567/puz8002 for conjugation into s. venezuelae .
a null mutant generated by double crossing over was identified by its apramycin - resistant and kanamycin - sensitive phenotype and named sv77 after confirmation by pcr using test primers listed in table s4 .
the mutant allele bldd::apr was moved into a new wt background by generalized transduction using the s. venezuelae - specific phage sv1 ( stuttard , 1979 ) . to prepare sv1-lysate ,
10 phage were added to 10 sv77 donor spores in 800 l pre - warmed ( 45c ) soft nutrient agar ( sna ) and poured onto difco nutrient agar plates containing 0.5% glucose , 10 mm mgso4 and 10 mm ca(no3)2 .
the plates were incubated at 30c overnight , then flooded with 2.5 ml difco nutrient broth ( dnb ) and incubated for 3 - 4 hr at room temperature . the phage - containing dnb soak - out was harvested and filtered through a 0.45 m filter to eliminate bacterial contamination .
for transduction of the bldd::apr allele , 10 phage particles harvested from the bldd::apr mutant strain sv77 , were mixed with 10 - 10 wt spores and incubated overnight on mym agar at room temperature before overlaying with apramycin for selection .
transduction of the bldd::apr allele was confirmed by pcr using test primers listed in table s4 , and the strain was named sv74 .
cyclic di - gmp capture compound experiments were performed as described previously ( nesper et al . , 2012 ) with minor modifications .
s. venezuelae cultures were grown in mym - tap supplemented with trace element solution until late transition phase for 24 hr at 30c and then pelleted by centrifugation for 10 min at 6,000 rpm .
the pellet was resuspended in lysis buffer ( 6.7 mm mes , 6.7 mm hepes , 200 mm nacl , 6.7 mm potassium acetate ( kac ) , ph 7.5 ) containing protease inhibitor and dnase i. cells were put through a french press four times at 18,000 psi and then centrifuged at 100,000 x g for 1 hr .
for the capture experiments , the protein concentration of the soluble fraction was determined using a uv / vis nanodrop spectrophotometer and 400 g protein were mixed with 10 m c - di - gmp capture compound and with 20 l 5 x capture buffer ( 100 mm hepes , 250 mm kac , 50 mm magnesium acetate ( mgac ) , 50% glycerol , ph 7.5 ) . 1 mm c - di - gmp was added to the control reaction and incubated for 30 min prior to capture compound addition .
the reaction volume was adjusted with h2o to 100 l and incubated for 2 hr at 4c in the dark on a rotary wheel .
after uv irradiation for 4 min in a caprobox , 50 l magnetic streptavidin beads and 25 l 5 x wash buffer ( 250 mm tris ph 7.5 , 5 m nacl , 0.1% n - octyl--glucopyranoside ) were added to the reaction and the mixture was incubated for 45 min at 4c on a rotary wheel .
the beads were then collected with a magnet and washed 6 times with 200 l wash buffer .
the beads were resuspended in 20 l sample buffer and run for 10 min on a 15% sds polyacrylamide gel after 10 min incubation at 95c .
the gel was stained using instantblue coomassie stain solution , and a 11 cm gel slice containing all captured proteins was excised for analysis by mass spectrometry .
dna fragments spanning the bldm ( 158 bp ) and whig ( 151 bp ) promoter regions of s. venezuelae were generated by pcr using oligonucleotides listed in table s4 and then 5 end - labeled using [ -p]-atp and t4 polynucleotide kinase .
the binding reactions were performed in bandshift buffer ( 10 mm tris ph 7.5 , 1 mm edta , 5% glycerol , 10 mm nacl , 1 mm mgcl2 ) in 20 l reaction mixture containing 0.6 m bldd and radiolabeled dna ( 8,000 c.p.m . ) as well as 0.5 g poly[d(i - c ) ] as nonspecific competitor dna .
when appropriate , increasing amounts of c - di - gmp ( 0.25 1.75 m ) were added to the mixture .
the reaction samples were incubated for 20 min at room temperature followed by electrophoresis on a 5% polyacrylamide gel in 0.5 x tbe ( tris - borate - edta ) buffer at 80v for 105 min .
chromatin immunoprecipitations were performed as previously described ( bush et al . , 2013 ) , except that an anti - bldd polyclonal antibody was used and pulled down with protein a - sepharose beads . library construction ,
sequencing and chip - seq data analyses were all carried out as previously described ( bush et al . , 2013 ) .
the his6-bldd - dbd and his6-bldd - ctd proteins were dialyzed into crosslinking buffer ( 100 mm nah2po4 , 150 mm nacl , ph 8) and then incubated at room temperature for 30 min in 20 l reaction samples containing 10 m protein , 1 mm disuccinimidyl suberate ( dss ) in dimethylsulfoxide ( dmso ) , and c - di - gmp as indicated .
the reaction was stopped by adding 50 mm tris ph 8 and incubation for 15 min followed by addition of sds sample buffer and heating to 95c for 10 min .
samples were separated on a 15% sds polyacrylamide gel and visualized by coomassie staining . radiolabeled c - di - gmp was synthesized in vitro using [ -p]-gtp and the purified diguanylate cyclase pled as described ( paul et al . , 2004 ) . the dracala assays ( roelofs et al . , 2011 )
were performed using 2 g of his6-bldd or its n - terminally his - tagged domains that were incubated with 11 nm p - c - di - gmp in dgc buffer ( 250 mm nacl , 25 mm tris ph 8 , 10 mm mgcl2 , 5 mm -mercaptoethanol ) . for competition experiments ,
266 m cold c - di - gmp or gtp were added to the reaction .
after a 5 min incubation at room temperature , 5 l of the binding sample were spotted onto nitrocellulose membrane and the dried membranes were analyzed using a phosphorimager . for structural studies on the ctd ,
the regions encoding residues 80 - 166 ( s. venezuelae bldd ) and 80 - 167 ( s. coelicolor bldd ) were cloned into the pet15b vector and the proteins induced at 37c and purified via ni - nta chromatography .
the his - tags were removed from the proteins used for structural studies by thrombin cleavage .
crystals of the s. venezuelae bldd ctd-(c - di - gmp ) complex , which assumed the trigonal space group , p32 , were obtained using protein at 30 mg / ml and 1 mm c - di - gmp .
crystals were produced via the hanging drop vapor diffusion method and mixing the complex 1:1 with 28% peg 1500 , 100 mm sodium acetate , ph 5.5 .
the s. venezuelae bldd ctd contains no methionines and hence for phasing , leu92 and ile135 were substituted with methionines .
semet(l92m / i135 m ) bldd ctd was expressed using the methionine inhibitory pathway and the protein purified and crystallized with c - di - gmp as per the wt ctd .
the selenomethionine - substituted l92m / i135 m protein crystallized in the wild - type protein p32 space group .
a second crystal form of the s. venezuelae bldd ctd-(c - di - gmp ) complex was grown with protein at 20 - 40 mg / ml and 1 mm c - di - gmp using 20% peg 2000 monomethyl ether , 100 mm mes , ph 6.0 , as a crystallization reagent and took the orthorhombic space group p21212 .
the third crystal form of the s. venezuelae bldd ctd-(c - di - gmp ) complex was produced with protein at 10 mg / ml and 1 mm c - di - gmp using 1.2 m sodium / potassium phosphate , 50 mm citrate ph 5.6 .
crystals were obtained for the s. coelicolor bldd ctd-(c - di - gmp ) complex using 25 mg / ml protein , 1 mm c - di - gmp and mixing the complex 1:1 with a reservoir comprised of 1.4 m sodium / potassium phosphate , 100 mm hepes ph 7.5 .
multiple wavelength anomalous diffraction ( mad ) data were collected for a semet(l92m / i135 m ) s. venezuelae bldd ctd-(c - di - gmp ) crystal to 2.28 resolution at als ( advanced light source , berkeley , ca , usa ) beamline 8.3.1 ( table s1 ) .
the data were processed using mosflm and the heavy atom substructure was obtained via solve ( terwilliger and berendzen , 1999 ) .
phenix was used for final phasing and density modification ( adams et al . , 2010 ) .
the crystal contains 12 protein molecules in the asymmetric unit ( asu ) and each of the six dimers is glued together by four c - di - gmp molecules .
the six c - di - gmp complexed dimers are essentially identical ( figure 4d ) .
final refinement was done using a data set collected to 1.95 resolution for a semet(blddl92 m ) ctd-(c - di - gmp ) crystal .
a wt data set was also collected to 2.2 resolution and the structure was identical to the l92 m and l92m / i135 m structures .
the final 1.95 resolution - structure contains residues 84 - 161 for each of the 12 subunits and 24 c - di - gmp molecules ( table s2 ) .
data were collected to 1.75 , 2.25 and 2.33 resolution for the s. venezuelae and s. coelicolor bldd ctd-(c - di - gmp ) c2221 forms and the s. venezuelae bldd ctd-(c - di - gmp ) p21212 crystal form , respectively , and the structures solved by molecular replacement ( mr ) .
the s. coelicolor bldd ctd and s. venezuelae bldd ctd c2221 crystal forms contain a ctd dimer and four c - di - gmp molecules in the asu and the s. venezuelae bldd c - domain-(c - di - gmp ) p21212 crystal form contains 10 subunits ( five dimers ) , and 20 c - di - gmp molecules .
the structures were solved by mr using the program phaser ( mccoy et al . , 2007 ) .
the topology of every bldd ctd is 1 ( residues 84 - 88)-1 ( residues 89 - 93)-2 ( residues 98 - 113)-2 ( residues 120 - 124)-3 ( residues 128 - 136)-4 ( residues 140 - 149)-5 ( residues 154 - 160 ) .
the bldd dna binding domain ( bldd dbd ) , encoding residues 1 - 79 , was cloned into pet15b , expressed in e. coli bl21(de3 ) and the protein purified via ni - nta chromatography .
prior to crystallization , the hexa - his tag was removed via thrombin cleavage and the protein further purified by size exclusion chromatography .
crystals were grown by mixing the protein ( 40 mg / ml ) 1:1 with a reservoir consisting of 35% peg 400 , 0.1 m mgcl2 and 0.1 m tris ph 7.5 .
x - ray intensity data were collected to 2.8 resolution at als beamline 8.3.1 and processed with mosflm .
the rsym and i/(i ) for the data are 11.3% ( 38.4% ) and 11.4 ( 4.3 ) , respectively , where the values in parentheses indicate data from the highest resolution shell .
the structure , which contains 3 subunits in the asu ( 2 subunits form a dimer and crystal symmetry generates a second dimer ) , was solved with phaser using a single s. coelicolor dbd subunit ( pdb code 2ewt ) as the search model .
the final model contains residues 3 - 71 of each subunit and was refined using phenix to final rwork / rfree values of 23.7%/28.9% , respectively ( adams et al . , 2010 ) .
crystals of the full - length ( fl ) s. venezuelae bldd-(c - di - gmp)-21-mer complex were grown by using protein in which the n - terminal hexa - his tag had been cleaved and incubated with 1 mm c - di - gmp ( final concentration ) .
this bldd-(c - di - gmp ) solution was mixed in a 1:1 molar ratio of bldd dimer to 21-mer dna duplex ( 5-cccctcacgctgcgtgacggg-3 , with the canonical bldd box underlined , annealed to its complementary oligodeoxynucleotide ) for crystallization trials .
crystals were grown by mixing the protein - dna complex 1:1 with the crystallization solution composed of 100 mm sodium citrate tribasic / citric acid ph 4.0 and 200 mm ammonium sulfate .
the crystals take the trigonal space group , p3221 , with a = b = 114.0 , c = 95.2 and contain a bldd dimer-(c - di - gmp)-21-mer duplex in the asu .
data were collected to the limiting resolution of 4.5 . the rsym = 9.0% ( 86.0% ) and i/(i ) = 5.8 ( 1.8 ) , where the values in parentheses are for the highest resolution shell .
first , bldd dna binding domain - dna complex models were constructed based on the sinr - dna ( pdb code 3zkc ) or repressor - dna ( pdb code 1lmb ) complex structures and used in the mr program phaser ( mccoy et al . , 2007 ) .
this solution was then used as a static model with the ctd-(c - di - gmp ) dimeric structure as a search model .
, the structure was subjected to rigid body refinement only ( rwork / rfree = 33.0%/38.9% , respectively ) . to examine the flexibility of the linker region that connects the bldd dbd and ctd ,
specifically , the accessibility of residue glu80 , which is the only acidic residue in the bldd linker region , was determined by the ability of the glu - c protease to cleave after this residue . in these experiments ,
endo - glu - c ( 100 units / ml ) was a diluted 50 fold into samples of 1 ) fl bldd ( 1 mg / ml ) , 2 ) fl bldd-21-mer dna ( 1 mg / ml protein with 100 m dna ) or 3 ) fl bldd-(c - di - gmp)-21-mer ( 1 mg / ml protein with 100 m 21-mer dna and 1 mm c - di - gmp ) .
the proteolysis buffer was 50 mm sodium phosphate , ph 7.5 and the final protein concentration of each sample was 4 mg / ml .
the samples shown in figure s6d were taken at a time point of 4 hr .
notably , proteolysis of each sample was identical , demonstrating that ligand binding of neither cognate dna nor c - di - gmp by bldd affect the accessibility of the linker region , which remains flexible and unstructured .
size exclusion chromatography experiments were carried out using a hiload 16/600 superdex 75 pg column .
5 mg of either the wild - type bldd ctd or the quadruple mutant were loaded onto a column that had been pre - equilibrated with 150 mm nacl , 5% glycerol , 20 mm tris hcl ph 7.5 and 3 m c - di - gmp .
the samples were run and eluted with the same c - di - gmp containing buffer .
the elution volume was plotted against a standard curve to determine the relative molecular weights of the samples .
the standard curve was determined using cytochrome c ( 12 kda ) , carbonic anhydrase ( 29 kda ) and albumin ( 66 kda ) .
cryo - sem was performed as previously described ( bush et al . , 2013 ) . when induced in e. coli bl21(de3 ) at 37c , bldd ctd samples contained little to no c - di - gmp contamination , which was supported by a280/a260 values of the purified protein , which were approximately 1.6 .
however , to ensure that there was no endogenous c - di - gmp present in samples used for binding affinity measurements , the bldd ctd was expressed and purified in sf9 insect cells . for these studies , a gene encoding the same s. venezuelae bldd ctd region that was expressed in e. coli was codon optimized for expression in insect cells ( genscript , piscataway , nj , usa ; http://www.genscript.com ) and subcloned into the expression vector f1 , which was transfected into the dh10bac strain for generation of the recombinant bacmid .
rbacmids were then transfected in sf9 insect cells with cellfectin ii and the cells incubated in sf-900 ii sfm for 56 days before harvest .
the supernatant was collected for the p1 viral stock and p2 was amplified for later infection .
sf9 cells expressing bldd ctd were harvested at 72 hr post infection and cells were lysed into 25 mm tris ph 7.5 , 300 mm nacl and 5% glycerol with protease inhibitors .
the protein was purified from the supernatant by ni - nta followed by size exclusion chromatography and was > 90% pure as assessed by sds - page analysis . to measure binding , 2-o-(6-[fluoresceinyl]aminohexylcarbamoyl)-cyclic diguanosine monophosphate ( 2-fluo - ahc - c - di - gmp ) ,
this molecule is conjugated via a 9 atom spacer to one of the 2 hydroxyl groups of the c - di - gmp , hence meeting the structural requirement for bldd binding that only one 2 hydroxyl group be unmodified and the other available for the interactions observed in the bldd-(c - di - gmp ) structures ( figure 5 ) .
binding was carried out at 25c in a buffer of 150 mm nacl and 25 mm tris - hcl ph 7.5 , which contained 1 nm 2-fluo - ahc - c - di - gmp .
increasing concentrations of bldd ctd were titrated into the reaction mixture to obtain the binding isotherms .
after each addition of protein the reaction sample was incubated for 30 min to ensure equilibrium had been reached .
c - di - gmp binding to bldd appears to have the characteristics of a high affinity / slow binding ligand .
the resulting data were plotted using kaleidagraph and curves were fitted to deduce binding affinities . to determine the binding stoichiometry of the bldd ctd-(c - di - gmp ) complex , the same fp binding conditions were used but the total concentration of c - di - gmp ( c - di - gmp + 2-fluo - ahc - c - di - gmp ) was 25 m , 10-fold above the kd of 2.5 m ensuring stoichiometric binding .
close inspection of the binding isotherm revealed the possibility that there are two nearly identical binding events ( likely corresponding to the binding of each intercalated c - di - gmp dimer ) .
the graph of the resulting data shows a linear increase in the observed mps until saturation of the binding sites , after which the line is flat .
importantly , the final inflection occurs at a bldd monomer concentrations of 12 m , which , when divided by the concentrations of c - di - gmp ( 25 m ) , indicates a stoichiometry of two ctd protomers per four c - di - gmps .
as anticipated from a purely chemical complementarity - orientated argument , fp studies carried out in the same buffer also revealed that wild - type bldd ctd does not bind to c - di - amp .
in addition , the bldd dgr - x8-dqdr ctd mutant did not bind the c - di - gmp probe .
n.t . designed , performed , and interpreted experiments , created figures , and wrote the paper . m.a.s . designed , performed , and interpreted experiments , created figures , and wrote the paper .
r.g.b . designed and interpreted experiments and wrote the paper . m.j.b . designed and interpreted experiments and wrote the paper | summarythe cyclic dinucleotide c - di - gmp is a signaling molecule with diverse functions in cellular physiology . here
, we report that c - di - gmp can assemble into a tetramer that mediates the effective dimerization of a transcription factor , bldd , which controls the progression of multicellular differentiation in sporulating actinomycete bacteria .
bldd represses expression of sporulation genes during vegetative growth in a manner that depends on c - di - gmp - mediated dimerization .
structural and biochemical analyses show that tetrameric c - di - gmp links two subunits of bldd through their c - terminal domains , which are otherwise separated by 10 and thus can not effect dimerization directly .
binding of the c - di - gmp tetramer by bldd is selective and requires a bipartite rxd - x8-rxxd signature .
the findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions . |
a 56-year - old man was observed in the emergency room ( er ) 4050 min after the onset of acute de - novo stabbing , pulsating and diffuse headache with subsequent appearance ( within few minutes ) of dysarthria , postural instability with retropulsion , mild objective vertigo and vomiting , acral paresthesias of upper limbs , minimal confusional state without loss of consciousness or seizures .
neurological examination was relevant for reagent anisocoria ( left smaller than right ) , slight speech disorder ( dysarthria ) , retropulsion , moderate bilateral incoordination at the index - nose test , absence of fever and meningeal signs .
blood pressure ( bp ) was unstable with recurrent hypertensive crises ( 200/120 mmhg ) during observation , in the absence of definite previous history of hypertension as well as of diabetes , headache , or cardiovascular problems .
electrocardiographic monitoring excluded paroxysmal arrhythmias , while cardiac enzymes and creatine - kinase were negative . in the er , he was started on intravenous ( i.v . )
nimodipine ( 2 mg / hour ) with progressive normalization of bp values within 4 h. urgent brain computerized tomography ( ct ) was negative for hemorrhage and focal lesions . considering the unstable bp and the low nihss ( national institutes of health stroke scale ) score ( =3 ) at presentation , i.v .
thrombolysis for possible brainstem ischemia was excluded , and the patient was put on antiplatelet therapy with salicylic acid ( i.v .
250 mg for 24 h , followed by oral administration ) as for a minor ischemic events . under i.v .
salicylic acid , headache severity gradually decreased over the next 3 h , leaving only a slight diffuse , pulsating pain which remitted over the next 6 h. all the neurological signs and symptoms had completely recovered within 12 h. in order to investigate the vertebro - basilar ( vb ) district , the patient also underwent urgent brain ct - angiography with maximum intensity projection ( mip ) , multiplanar ( mp ) and three - dimensional ( 3d ) reconstructions , with the detection of left persistent primitive hypoglossal artery ( ppha ) as a large vessel originating from the internal carotid artery ( ica ) at the c2 vertebral level , entering the posterior cranial fossa through an enlarged hypoglossal canal and thus joining the lower portion of the basilar artery ( fig . 1 ) .
the study was then completed through 3d volume - rendering ( vr ) reconstructions for a better definition of the anatomical details ( fig . 1 ) .
collateral findings were represented by bilateral vertebral artery hypoplasia together with a small saccular aneurysm ( not - surgical ) of the anterior communicating artery , confirmed by digital subtraction angiography ( dsa ) which revealed no additional vascular abnormalities .
brain magnetic resonance ( mr ) with mr - diffusion weighted imaging ( mr - dwi ) was performed 24 h after onset of symptoms and did not show recent ischemic lesions .
the electroencephalogram performed 12 h after admission did not record epileptic / focal abnormalities .
uncommon causes of stroke ( thrombophilia , patent foramen ovale and other relevant metabolic risk factors for stroke ) were excluded during the course of the observation.fig .
1a axial brain ct - angiography , b , c mip and 3d - reconstruction ct - angiography showing left ppha ( arrows ) .
d three - dimensional - vr ct - angiography showing ppha ( arrow ) entering the posterior cranial fossa through an enlarged hypoglossal canal and thus joining the lower portion of the basilar artery , and in e ppha ( arrow ) as a large vessel originating from the internal carotid artery at the c2 vertebra level a axial brain ct - angiography , b , c mip and 3d - reconstruction ct - angiography showing left ppha ( arrows ) .
d three - dimensional - vr ct - angiography showing ppha ( arrow ) entering the posterior cranial fossa through an enlarged hypoglossal canal and thus joining the lower portion of the basilar artery , and in e ppha ( arrow ) as a large vessel originating from the internal carotid artery at the c2 vertebra level the patient was then discharged with ramipril ( 5 mg per day ) plus oral salicylic acid ( 100 mg per day ) and at the 6-month control no further episode of headache with or without neurological symptoms was reported .
headache associated to vb signs and symptoms , once excluded critical conditions , could suggest at least four possible diagnoses : ( 1 ) basilar - type migraine , ( 2 ) posterior reversible encephalopathy syndrome ( pres ) , ( 3 ) headache attributed to benign ( or reversible ) angiopathy of the central nervous system , and ( 4 ) headache attributed to transient ischemic attack ( tia ) or minor stroke , whose diagnostic criteria are summarized in table 1.table 1diagnostic criteria for basilar - type migraine , posterior reversible encephalopathy syndrome ( pres ) , headache attributed to benign ( or reversible ) angiopathy of the central nervous system , and headache attributed to transient ischemic attack ( tia)basilar - type migraine migraine with aura symptoms clearly originating from the brainstem and/or from both hemispheres simultaneously affected , but no motor weakness(a ) at least 2 attacks fulfilling criteria b
d(b ) aura consisting of at least two of the following fully reversible symptoms , but no motor weakness : dysarthria , vertigo , tinnitus , hypacusia , diplopia , visual symptoms simultaneously in both temporal and nasal fields of both eyes , ataxia , decreased level of consciousness , simultaneously bilateral paraesthesias(c ) at least one of the following : ( 1 ) at least one aura symptom develops gradually over 5 min and/or different aura symptoms occur in succession over 5 min ( 2 ) each aura symptom lasts 5 and 60 min(d ) headache fulfilling criteria b
d for 1.1 migraine without aura begins during the aura or follows aura within 60 min(e ) not attributed to another disorderposterior reversible encefalophathy syndrome ( pres ) acute or subacute neurologic presentation of headeache , nausea , vomiting , altered mental function , seizures , stupor , visual disturbancesradiological hallmarks : reversible bilateral subcortical and cortical edema with a predominantly posterior distribution ( parieto - occipital ) at fluid - attenuated inversion recovery ( flair ) mri imagingheadache attributed to benign ( or reversible ) angiopathy of the central nervous system ( a ) diffuse , severe headache of abrupt or progressive onset , with or without focal neurological deficits and/or seizures and fulfilling criteria c and d(b ) strings and beads
appearance on angiography and subarachnoid hemorrhage ruled out by appropriate investigations(c ) one or both of the following : ( 1 ) headache develops simultaneously with neurological deficits and/or seizures ( 2 ) headache leads to angiography and discovery of strings and beads
appearance(d ) headache ( and neurological deficits , if present ) resolves spontaneously within 2 monthsheadache attributed to transient ischemic attack ( tia ) ( a ) any new acute headache fulfilling criteria c and d(b ) focal neurological deficit of ischemic origin lasting < 24 h(c ) headache develops simultaneously with onset of focal deficit(d ) headache resolves within 24 h diagnostic criteria for basilar - type migraine , posterior reversible encephalopathy syndrome ( pres ) , headache attributed to benign ( or reversible ) angiopathy of the central nervous system , and headache attributed to transient ischemic attack ( tia ) according to the current criteria of the international classification of headache disorders2nd ed .
( ichd - ii ) , our case potentially fulfills only some of the diagnostic criteria for basilar - type migraine ( 1.2.6 ) : headache , dysarthria , vertigo ( with onset / worsening in few minutes ) , ataxia , decreased level of consciousness and simultaneous bilateral paraesthesias .
however , the neurological symptoms have lasted over 60 min and were associated with de - novo headache ( first episode ) , thus excluding the above hypothesis ( table 1 ) .
the second diagnosis we have considered , is pres , a rare and still poorly understood condition characterized by acute or subacute headache ( usually thunderclap type ) , nausea , vomiting , altered mental function , seizures , stupor and/or visual disturbances , with the radiological hallmarks of reversible bilateral subcortical and cortical edema with a predominantly posterior distribution ( parieto - occipital ) at fluid - attenuated inversion recovery ( flair)-mr sequences .
pres has been described in association with hypertensive encephalopathy , immunosuppressive and cytotoxic medications , puerperal eclampsia , collagen disease , renal failure , thrombotic thrombocytopenic purpura , human immunodeficiency virus infection , acute intermittent porphyria , and organ transplantation . in our case ,
the lack of the typical mr findings and the negative electroencephalogram tend to rule out this diagnosis .
headache attributed to benign ( or reversible ) angiopathy of the central nervous system ( ichd - ii
6.7.3 ) . in our case , despite the complete reversibility of all neurological signs , the lack of the radiological hallmarks strings and beads and the full headache recovery within 12 h , excludes this nosological entity ( table 1 ) .
the last possible and more suitable diagnosis is that of headache attributed to transient ischemic attack ( tia)ichd - ii 6.1.2
in fact , headache occurs frequently in patients with acute cerebrovascular disorders with a frequency according to different studies ranging from 7 to 65% .
several authors have stressed the greater incidence of headache among patients with ischemic events , particularly vb infarcts , due to the anatomical relationship between the posterior circulation and the trigeminal system .
although relatively less frequent during tia , sudden and acute headache of unknown cause , associated to dizziness or loss of balance , may represent a warning sign of vb failure .
the most common symptoms of extracranial vertebral artery involvement are represented by dizziness , blurred vision and imbalance , while vertigo is more typical of intracranial vertebral artery disease .
on the other hand , tia due to basilar artery failure produces , more frequently , dizziness , double vision , dysphagia , slurred speech and unilateral / bilateral weakness .
symptoms and signs due to tia ( either carotid or vb ) must resolve by definition within 24 h but sometimes they may be very brief lasting few minutes probably due to sudden and temporary bp failure . in this perspective , sacco et al .
have suggested to review the ichd - ii diagnostic criteria for 6.1.2 in the agreement with the new definition of tia proposed by the american heart association study group .
accordingly , tia is now defined as a transient episode of neurological dysfunction caused by focal brain , spinal cord or retinal ischemia , without acute infarction thus reducing the importance of the traditional temporal criterion for the definition of tia ( duration < 24 h ) . as a consequence ,
brain - mr imaging becomes now the crucial recommended tool for tia diagnosis . besides the above conditions discussed in the differential diagnosis versus the most probable hypothesis of headache attributed to transient ischemic attack ( tia ) or minor stroke , we can also exclude ichd - ii
10.3.2 ( headache attributed to hypertensive crisis without hypertensive encephalopathy ) , due to the presence of long - lasting focal neurological signs .
in the present case , de - novo acute headache and transient ischemia in the posterior circulation are associated with the evidence of ppha , a rare vascular anomaly of the posterior circulation that ensures a pathological anastomosis between carotid and basilar system . at the intracranial level , four embryonic arteries ( trigeminal , otic , hypoglossal and proatlantal )
participate to the vb development : once completed , these vessels gradually regress , but in some cases they may persist . the detection is often fortuitous during angiographic examination .
the trigeminal artery is generally associated with disorders of the 3rd , 4th , 5th and 6th cranial nerves , while the otic artery may cause acoustic or facial nerves palsy .
the hypoglossal artery may lead to 12th nerve palsy , glossopharingeal neuralgia or vascular disorders in the posterior circulation .
it originates as a branch of the cervical part of the ica between c1 and c3 levels , passes through the hypoglossal canal and joins the lower portion of the basilar artery after a tortuous course .
consequently , any significant alteration of the blood flow in the proximal ica may affect the posterior circulation with possible reversible vb symptoms .
less frequent is usually associated to ischemic lesions . from a pathogenetic point of view
, it seems therefore reasonable to consider , in our case , the particular vulnerability of the posterior circulation due to the ppha , the associated vascular abnormalities , and its possible secondary , transient , hemodynamic failure during hypertensive phases .
our clinical picture may be classified as a real tia of the posterior circulation , also considering the complete clinical recovery within 24 h and the absence of ischemic lesions ( including lacunar events ) on mr investigations .
moreover , the headache presented by the patient , meets the ichd - ii diagnostic criteria for headache attributed to transient ischemic attack ( tia)subgroup 6.1.2 . in conclusion , the persistence of carotid - basilar anastomoses ,
although rare entities , must be considered not only in patients with isolated or combined cranial nerve deficit , but also in the presence of cerebrovascular events involving the vb system .
their association with other vascular abnormalities ( hypoplasia ) can confer increased vulnerability to other factors ( hypertension ) as in the present case . | we report the case of a 56-year - old man with acute onset of de - novo stabbing , pulsating and diffuse headache with subsequent appearance ( within few minutes ) of posterior fossa symptoms ( vomiting , postural instability , anisocoria , incoordination , dysarthria , retropulsion ) lasting 912 h. recurrent hypertensive crises were detected during the acute observation in the emergency room , even in the absence of previous history of hypertension .
once subarachnoid hemorrhage and focal lesions ( vascular and non - vascular ) were excluded , brain computerized tomography - angiography and digital subtraction angiography disclosed the presence of left persistent primitive hypoglossal artery with bilateral vertebral artery hypoplasia and a slight aneurysmal dilation of the anterior communicating artery .
brain magnetic resonance study performed 24 h after onset of symptoms was negative for recent ischemic lesions .
the clinical features of this rare vascular condition are discussed as a possible cause of magnetic resonance ( diffusion weighted imaging ) negative vertebro - basilar transient ischemic attack . |
almost 170 million people are infected with hepatitis c virus ( hcv ) globally , which accounts for 2 to 2.2 percent of world population .
the virus prevalence is various in different countries and has been reported between 0.1% and 12% .
the main routes of this virus transmission are through blood , sexual contacts and sharing syringe practiced in shooting galleries ( 1 ) . in iran
the drug abusers are the main source of infection ( 2 ) ; they transmit the infection through practicing unsafe sex , sharing syringes and using shared shaving razors .
the second important group in danger of gaining the infection are those who received blood before the start of blood screening program in 1995 ( 3 ) .
these include thalassaemia and hemophilliac patients ; the prevalence rate of infection in them is 11.8% and 53.3% respectively ( 3 ) .
hcv prevalence in general population in iran is estimated to be between 0.12% and 0.89% ( 4 ) .
many people around the world are infected by hepatitis b virus ( hbv ) . the world health organization ( who ) has estimated that almost two billion people have been infected with this virus globally and almost 350 million chronically infected people live worldwide ( 5 ) .
prevalence of this virus in southeast asia , china and some parts of africa is high and reaches 8% , while in developed countries this rate is lower than 2% ( 5 , 6 ) . according to the latest reports , the rate of hbv prevalence in iran has declined to less than two percent ( 7 , 8) .
hcv and hbv are two major causes of liver cirrhosis and hepatocellular carcinoma ( hcc ) ( 1 , 6 ) and the rate of hbv - hcv co - infection is different among hbv chronic patients , ranging from 4.54% to 0.7% , which complicates the process of management and treatment of infected people ( 9 , 10 ) .
human t - cell lymphotropic virus-1 ( htlv-1 ) was first discovered by poiesz in the lymphocytes of a patient with cutaneous t - cell lymphoma .
this virus , after a long period of incubation time , causes a particular type of lymphoma called adult t - cell lymphoma ( atl ) ( 11 , 12 ) .
this virus also causes a neurological disorder called tropical spasmatic parapheresis ( tsp ) ( 12 ) .
htlv-1 is highly prevalent in southwest of japan , caribbean basin and some parts of africa ( 13 ) . in iran
this virus is prevalent in northeast of iran ( khorasan province ) . in a study ,
the rate of htlv prevalence in volunteer blood donors of this province reported 0.77% ( 14 ) . in another study among thalassemia patients and healthy control subjects , the researchers found that the rate of htlv-1 infection among patients was 1.6% in comparison to 0.5% in healthy control subjects ( 15 ) .
the aim of this study was to assess the hbv , hcv , htlv-1 seroprevalence among individuals attending the endoscopy ward and also evaluating the association between some demographic criteria with the status of antibody against these viruses .
this descriptive cross sectional study was performed between 2009 and 2011on two hundred and nineteen individuals attending the endoscopy ward of taleghani hospital , tehran , iran .
blood samples of enrolled individuals were taken and a questionnaire containing demographic information was filled for each participant .
the study was approved by the institutional medical ethics committees of research center for gastroenterology and liver disease of shaheed beheshti university of medical sciences .
sera were separated from blood and were kept in -20 degree celsius freezer until the serologic tests were undertaken .
the antibodies against htlv-1 and hcv were assessed through enzyme linked immunosorbent assay method ( dia.pro italy ) . for detection of hepatitis b virus infection status , at first sera were tested for antibody against hepatitis b core antigen ( anti - hbc ab ) using elisa technique ( dia.pro ) and if the result were positive , then we would test the sera for hepatitis b surface antigen ( hbs ag ) ( dia.pro ) .
test results and information gained from questionnaires were entered into spss program ( spss inc , chicago , usa ) and all of statistical analysis was performed by it .
student s t - test was utilized to compare means of some continuous variable of two independent sample groups .
continuous variables are represented as mean standard deviation and other parameters as frequency and percentage .
a p - value of 0.05 or less was considered statistically significant and all reported p- values were two sided .
92 of them ( 42% ) were male and 127 ( 58% ) were female .
the mean age of the population was 39.87 16.47 years ; their age range was between 13 and 84 years old .
the mean age of male participants was 37.51 16.33 and the mean age of female participants was 42.97 16.98 .
there was no statistically significant relationship between gender and mean age ( p = 0.076 ) .
two subjects ( 0.9% ) had hbs antigen , a sign of active infection , while 24 of anti - hbc positive individuals ( 92.31% ) had cleared the infection ( table 1 ) .
the frequency of antibody against hbv , hcv and htlv-1 among participants *
hbc ab : antibody against hepatitis b core antigen , hbs ag : hepatitis b surface antigen , hcv ab : antibody against hepatitis c virus , htlv-1 ab : antibody against human t - cell lymphotropic virus-1 mean age of those subjects who had antibodies against hbc antigen was 48.88 17.46 , whereas the mean age of those negative for antibody was 38.7116.02 ; the difference in these two groups was statistically significant ( p = 0.003 ) .
fourteen individuals from 26 people with anti - hbc antibody were female and twelve were male .
there was no significant relationship between gender and anti - hbc antibody status ( p= 0.65 ) .
the two subjects who had hbs ag were a male 51 years old and a female 61 years old . because of the low frequency of positive subjects for htlv-1 antibody and lack of positive individuals for hcv antibody , we were not able to perform any statistical analysis for these patients .
however , the mean age of positive subjects for htlv-1 was 61.50 15.18 years and the negative individuals were 39.48 16.26 years .
viral hepatitis is one of major concerns of public health in countries like iran ( 16 ) .
htlv-1 infection plays an important role in etiology of many cancers including atl and gastric cancer ( 13 , 17 ) . in our study
we did not find any individual with anti - hcv antibody which was similar to the results achieved by moradi and colleagues ( 18 ) .
mohebbi and colleagues in another study found out that the rate of hcv infection was only 0.2% among pregnant women in western iran ( 10 ) .
it is an indicator that the rate of hcv infection in our country is low .
apparently our findings is in contradiction with the results of sarkari and colleagues ( 19 ) , but in that study studied subjects were from a high risk population .
results of this study showed that a considerable number of participants have had exposure to hbv infection ( 11.87% ) , but have cleared the infection .
there was a significant relationship between mean age of participants and anti - hbc ab status , an indicator of a high prevalence of hbv infection in the past .
farzadegan and colleagues reported the rate of hepatitis b antigen among iranians between 2.5 and 7.2 percent ( 20 ) . according to different reports three percent of iranians
. additionally we have to keep in mind that with increasing the age of a subject , one s chance of contracting the virus rises accordingly . in alavian and colleagues
review article , gender was mentioned as a risk factor for acquiring hbv infection ( 8) .
however , we did not find any association between gender and anti - hbc seropositivity .
only 0.9% of our studied population had hbs antigen ( an indicator of active infection ) .
this low rate can be an indicator of declining trend of hbv infection in iranian general population .
this observation can be accounted for by mass mandatory vaccination in children and voluntary vaccination in adults and the improved condition of sanitation in iran and increase of public knowledge about routes of hbv transmission in recent years .
however in high risk individuals the rate of hbv infection in these people is still high ( 22 , 23 )
current study shows that htlv-1 prevalence rate is 1.82% .
abbaszadegan and colleagues reported the htlv-1 seroprevalence rate in 2003 in khorasan province 0.77% ( 14 ) .
tarhini and colleagues reported that in the years 2004 , 2005 and 2006 the rate of htlv-1 prevalence has declined to 0.5% , 0.44% and 0.42% , respectively ( 24 ) .
our study shows that the rate of infection in our studied population is higher in comparison to the previous studies .
however our study is in accordance with the results of study performed by arjmand and colleagues on iranian organ donors that reported the rate of htlv-1 prevalence 1.87% ( 25 ) .
results of this study are in accordance with the results achieved by heidari and colleague in which they studied the prevalence of blood - borne infections in cardiology ward of a hospital in mashahd ; the rate of hcv , hbv and htlv-1 infections were 0.3 , 2.37 and 2.59 percent , respectively ( 26 ) . in a study in west
africa the researchers found out that the rate of anti - hbc ab , hbs antigen , hcv and htlv-1 in two separate regions were 69.6/76.4 , 14.3/17.3 , 2.2/1.5 and 1.4/0.5 percent respectively ( 27 ) . in another study among immigrants to spain
, the researchers found out that the rate of hbv , hcv and htlv-1 prevalence was 4.1 , 2.9 and 0.8 percent respectively ( 28 ) .
the relatively high htlv-1 seroprevalence in compared to previous studies is alarming . in the light of achieved results we suggest that in addition to northeastern section of the country , the screening for htlv-1 become mandatory for other parts of iran .
this practice is being implemented in japan , a country with a high rate of htlv-1 seropositivity ( 29 ) . | aimthis study was designed to evaluate the frequency of antibody against these viruses in individuals attending the endoscopy ward of taleghani hospital tehran , iran.backgroundblood-borne viruses such as hepatitis b and hepatitis c virus and htlv-1 virus are among the world s public health problems .
hepatitis viruses cause liver problems and htlv-1 infection can lead to adult t - cell lymphoma ( atl).patients and methodsblood samples of 219 individuals attending the endoscopy ward of taleghani hospital between years 2009 - 2011 were collected . a questionnaire containing demographic data
was completed for each subject .
blood samples were tested for antibody against htlv-1 , hcv and hbc by elisa ( dia.pro italy ) . in case of positive results for anti - hbc , samples were also tested for hbs ag antigen.resultsninety two subjects were male and 127 were female .
mean age of the population was 39.87 16.47 .
none of the subjects had anti - hcv antibody , while 4 of them had anti - htlv-1 antibody and 26 anti - hbc antibody ; which only two of these individuals had hbs antibody.conclusionthe results of this study show that frequency of anti - hcv and anti - htlv-1 antibodies are very low , while the frequency of anti - hbc was higher in the population .
since htlv-1 is the causative agent of a type of blood cancer , it seems that screening of donated bloods in this region should be considered . |
hemodynamically significant stenosis of the subclavian artery usually presents with symptoms of upper limb ischemia on the ipsilateral side as the lesion .
it may also present as subclavian steal syndrome with symptoms of vertebro - basilar insufficiency as a result of retrograde flow in the ipsilateral vertebral artery .
the most common cause of subclavian artery stenosis is atherosclerosis but other causes include congenital abnormalities such as arteria lusoria ( aberrant subclavian artery ) or right sided aortic arch that can cause compression of the right subclavian artery leading to congenital subclavian steal syndrome , , .
an endovascular approach is attempted before proceeding to open subclavian artery revascularization as it is a less invasive procedure .
a retrospective study on the long - term outcome of this endovascular intervention concluded that there was high primary success with satisfactory outcomes beyond 10 years .
the study conducted by stone et al . compared endovascular treatment and open surgery in subclavian occlusive disease .
the study concluded that endoluminal therapy for subclavian disease is effective and safe ; however , open surgery still carries a better long - term durability and should be the preferred approach in low - risk patients .
symptomatic patients who failed endovascular treatment or subsequent loss of patency by stent occlusion should be considered for surgical revascularization .
the carotid subclavian bypass is a safe and commonly used surgical procedure when endovascular intervention has failed .
a retrospective study analysing outcomes of common carotid - subclavian artery bypass versus transposition of subclavian artery on the common carotid artery concluded that the latter should be considered the first line surgical management of proximal subclavian artery lesions .
the study showed the six - year patency rate was one hundred percent for transposition of subclavian artery on the common carotid artery and sixty - six percent for carotid subclavian bypass . in this case series , we describe different modalities of intervention in four patients presenting with ischaemic symptoms of subclavian stenosis .
a 71 year old female presented as an emergency with a ten day history of a cold left arm following a fall onto her left side . on examination , her left upper limb was cold , pale and pulseless .
symptoms initially improved but she had ongoing pain in her left upper limb and developed dry gangrene of her left thumb .
she underwent stenting of the subclavian artery which restored luminal patency and relieved her symptoms .
1b ) . where appropriate , angioplasty and stenting is the first line treatment for subclavian artery stenosis .
a 59 year old male smoker was referred to vascular outpatient clinic with a history of a cold left upper limb over the last few years .
mra showed an occlusion of the proximal left subclavian artery extending to the origin of the left vertebral artery ( fig .
angioplasty and stenting was attempted on two occasions but the procedures were terminated because it was not possible to cross the occlusion . following multidisciplinary team discussion , it was decided to offer a carotid subclavian bypass with transposition of the subclavian artery onto the common carotid artery . via a transverse supraclavicular incision ,
the left subclavian artery , left thyrocervical trunk and left vertebral arteries were dissected and controlled .
the left subclavian artery was clamped just distal to the occlusion , proximal to the origin of the left vertebral artery , and transected .
a longitudinal arteriotomy was made in the left common carotid artery and an end - to - side anastomosis was performed between the left subclavian artery and left common carotid artery .
after 6 months a mra showed that the transposition of the left subclavian artery to the left common carotid artery was successful , with slight narrowing at the origin of the subclavian artery .
the left vertebral artery was patent and the origin of the left subclavian artery is now a small blind ending trunk ( fig .
the transposition of the subclavian onto the carotid artery technique was possible because the lesion was proximal to the origin of the vertebral artery and the carotid artery was not diseased .
transposition of the subclavian artery to the carotid artery is most commonly performed in preparation for stenting of aneurysms of the thoracic aorta . a 64 year old female smoker with severe chronic obstructive pulmonary disease and peripheral vascular disease requiring lower limb vascular stents
was found to be complaining of left upper limb claudication at a vascular outpatient review appointment .
duplex ultrasonography showed stenosis in the mid left subclavian artery with reversed flow in the left vertebral artery , in keeping with subclavian steal .
multidisciplinary team advice was to offer carotid subclavian artery bypass . via a transverse left supraclavicular incision
proximal and distal control of the common carotid artery and the subclavian artery was gained .
initial angiograms obtained through the femoral artery in the groin showed a tight stenosis at the origin of the subclavian artery .
the duplex repeat angiogram confirmed an occlusion of the left subclavian stent and she underwent further angioplasty and insertion of a stent .
unfortunately , the stent blocked again for the second time , and a decision made to carry out a bypass rather than perform repeat radiological re - intervention .
she therefore underwent a carotid to axillary bypass . via a longitudinal incision along the medial aspect of the left sternocleidomastoid muscle ,
a six millimetre supported ptfe graft was tunneled under the sternocleidomastoid muscle and over the clavicle and anastomosed . in cases where previous stenting of the subclavian artery complicates any anastomosis involving the subclavian artery , carotid - axillary bypass is a good alternative .
a 71 year old female presented as an emergency with a ten day history of a cold left arm following a fall onto her left side . on examination , her left upper limb was cold , pale and pulseless .
symptoms initially improved but she had ongoing pain in her left upper limb and developed dry gangrene of her left thumb .
she underwent stenting of the subclavian artery which restored luminal patency and relieved her symptoms .
1b ) . where appropriate , angioplasty and stenting is the first line treatment for subclavian artery stenosis .
a 59 year old male smoker was referred to vascular outpatient clinic with a history of a cold left upper limb over the last few years .
mra showed an occlusion of the proximal left subclavian artery extending to the origin of the left vertebral artery ( fig .
angioplasty and stenting was attempted on two occasions but the procedures were terminated because it was not possible to cross the occlusion .
following multidisciplinary team discussion , it was decided to offer a carotid subclavian bypass with transposition of the subclavian artery onto the common carotid artery . via a transverse supraclavicular incision ,
the left subclavian artery , left thyrocervical trunk and left vertebral arteries were dissected and controlled .
the left subclavian artery was clamped just distal to the occlusion , proximal to the origin of the left vertebral artery , and transected .
a longitudinal arteriotomy was made in the left common carotid artery and an end - to - side anastomosis was performed between the left subclavian artery and left common carotid artery .
after 6 months a mra showed that the transposition of the left subclavian artery to the left common carotid artery was successful , with slight narrowing at the origin of the subclavian artery .
the left vertebral artery was patent and the origin of the left subclavian artery is now a small blind ending trunk ( fig .
the transposition of the subclavian onto the carotid artery technique was possible because the lesion was proximal to the origin of the vertebral artery and the carotid artery was not diseased .
transposition of the subclavian artery to the carotid artery is most commonly performed in preparation for stenting of aneurysms of the thoracic aorta .
a 64 year old female smoker with severe chronic obstructive pulmonary disease and peripheral vascular disease requiring lower limb vascular stents was found to be complaining of left upper limb claudication at a vascular outpatient review appointment .
duplex ultrasonography showed stenosis in the mid left subclavian artery with reversed flow in the left vertebral artery , in keeping with subclavian steal .
multidisciplinary team advice was to offer carotid subclavian artery bypass . via a transverse left supraclavicular incision
proximal and distal control of the common carotid artery and the subclavian artery was gained .
a 56 year old female smoker presented acutely with left arm numbness and pain . there were no palpable pulses .
initial angiograms obtained through the femoral artery in the groin showed a tight stenosis at the origin of the subclavian artery .
the duplex repeat angiogram confirmed an occlusion of the left subclavian stent and she underwent further angioplasty and insertion of a stent .
unfortunately , the stent blocked again for the second time , and a decision made to carry out a bypass rather than perform repeat radiological re - intervention .
she therefore underwent a carotid to axillary bypass . via a longitudinal incision along the medial aspect of the left sternocleidomastoid muscle ,
a six millimetre supported ptfe graft was tunneled under the sternocleidomastoid muscle and over the clavicle and anastomosed . in cases where previous stenting of the subclavian artery complicates any anastomosis involving the subclavian artery , carotid - axillary bypass is a good alternative .
haemodynamically significant stenosis of the subclavian artery may present with symptoms of upper limb ischemia , subclavian steal syndrome or , more rarely , coronary steal syndrome .
we have described four methods of restoring blood flow to the upper limb in cases of symptomatic subclavian artery stenosis .
it is widely accepted that endovascular stenting of the subclavian artery should be first line treatment for this condition .
angioplasty can however , can cause intraluminal hyperplasia and the re - stenosis rates are higher than for extrathoracic surgical revascularisation .
extrathoracic surgical revascularisation becomes necessary when endoluminal measures fail , or when anatomical variations make it more technically difficult .
transposition of the subclavian artery onto the carotid artery is suitable if there is proximal stenosis of the subclavian artery , allowing for mobilisation of the subclavian artery distal to the stenosis . in the management of proximal subclavian artery stenosis it is safe and re - occlusion rates are low .
re - occlusion rates are lower than those for carotid - subclavian artery bypass using a graft .
this technique has been used to treat subclavian steal due to a congenital aberrant right subclavian artery .
another common reason for doing this operation is in preparation for stenting of thoracic aortic aneurysms , to reduce the risk of cerebral ischemia , .
carotid - subclavian artery bypass using a prosthetic graft is considered as a safe surgical intervention with mortality rates of 03% and stroke risk of 05% .
these rates are comparable with those for transposition of the subclavian artery onto the carotid artery .
a wide range of grafts may be used , including dacron , autologous vein and ptfe .
have demonstrated the safety and effectiveness of carotid - subclavian bypass in their analysis of 287 patients treated for subclavian artery stenosis in this way .
carotid - subclavian bypass has also been used in repair of aberrant right subclavian artery aneurysm following endoluminal aortic stent graft exclusion .
it has the advantage of avoiding the area of stenosis in cases where the subclavian artery has extensive disease and damage from previous angioplasty and stenting .
it is important to consider the options available when considering surgical management of subclavian artery stenosis .
in addition to atherosclerosis and damage from previous endovascular treatments , anatomical variations such arteria lusoria may complicate surgical decisions .
it is important for the surgeon to have a thorough understanding of the anatomy and aetiology while considering surgical options .
this original case series demonstrates some of the treatment options available to vascular surgeons when managing symptomatic subclavian artery disease .
rs , jh and ljw collected data , performed a literature review and wrote the paper .
| highlightscase presentation of a patient treated with subclavian artery stenting.case presentation of a patient treated with transposition of the left subclavian artery onto the left common carotid artery.case presentation of a patient treated with carotid- subclavian artery bypass with a ptfe graft.case presentation of a patient treated with carotid to axillary bypass.discussion and literature review of methods and indications of treatment of subclavian artery occlusive disease . |
ampulla cardiomyopathy , i.e. takotsubo cardiomyopathy ( tc ) , is a cardiac syndrome with acute onset defined by chest symptoms and elevated st segment on the echocardiogram without significant narrowing of the coronary arteries .
takotsubo , the name of a traditional japanese octopus pot with a wide base and tapered top . since tc mimics coronary artery disease , early diagnosis of it is very important .
we report a case of tc after surgery for gallbladder and common bile duct stones .
an 85-year - old naturally nervous woman was admitted to the hospital for obstructive jaundice .
she was not a smoker , had no past medical history including coronary heart diseases and no special family history . on admission ,
1a ) or vital signs . ercp ( endoscopic retrograde cholangiopancreatography ) and echography demonstrated gallstones and common bile duct stones ( fig .
laboratory data were as follows : wbc 21,600/l , platelets 95,000/l , t.bil 7.9 mg / dl , ast 98
u / l , alt 139 u / l , crp 16.8 mg / dl , cea 2.4 ng / ml , and ca19 - 9 2 u / ml .
ultrasonography revealed many small gallbladder stones and ercp revealed 4 stones ( 520 mm in size ) in the common bile duct .
est ( endoscopic sphincterotomy ) was unsuccessful because those stones were large and stuck in the common bile duct . under a diagnosis of choledocolithiasis and cholecystitis with moderate icterus
, the patient was first treated conservatively , with bile drainage by enbd ( endoscopic nasobiliary drainage ) tube and administration of antibiotics . though laboratory data showed sepsis ( platelets 45,000/l ) on the next day , she successfully recovered in 2 weeks ( platelets 110,000/l ) , when cholecystectomy and choledocholithotomy were successfully performed . just after the operation , on the day of surgery , the ecg suddenly demonstrated st - t elevation in leads ii ,
laboratory examination revealed slight elevation of cpk ( 367 mg / dl ) and cpk - mb ( 16 mg / dl , 4% ) although troponin - i was normal and the echocardiogram showed asynergy with apical akinesis , basal hyperkinesis and decreased contraction of the heart ( lvdd / ds = 47/29 mm ) ( fig .
takotsubo. her cvp was constantly 79 cm h2o , and there were no symptoms of heart failure .
she recovered from sepsis in a week , and her pressure gradient was low , although no signs of cardiac failure were detected during the postoperative course .
the c - tube was removed on the 4th postoperative day . on day 2 after the operation , the ecg showed inverted t waves in leads v2-v5 ( fig .
tc was first reported by dote et al . as a cardiac syndrome with acute onset , reversible ventricular wall motion disorder with chest symptoms , electrocardiographic changes with st elevation and/or depression , and minimal myocardial enzymatic release mimicking acute coronary syndrome but without angiographical stenosis of the coronary arteries .
though most cases of tc have been reported in japan , recently tc has come to be focused on in the us as well .
spect imaging has been reported as a method to assess tc , and tc might represent a stunned myocardium caused by disturbance of the coronary microcirculation .
though the pathophysiological mechanism of tc remains unclear , some hypotheses have been offered for it .
reported that tc might be related to a disturbance of cardiac sympathetic innervation . on the other hand , akashi et al . insisted that catecholaminergic or adrenoceptor - hyperactive cardiomyopathy might be the cause of tc
moreover , tc should be considered in aged patients and in postmenopausal females who must undergo physically and mentally stressful treatment .
it is generally thought that - blockers might be effective for this syndrome , though they should be used carefully because of their various side effects .
it should be also reminded that like in the present case , no medication might be necessary with careful follow - up .
recently , tc has been reported in connection with stress in surgery or anesthesia . besides the present case ,
only one case of tc has been reported after hepatopancreatic- biliary surgery , although it might be suggested that similar cases are increasing . since it is impossible to predict the occurrence of tc , ecg changes
useful criteria such as mayo criteria should always be borne in mind as early detection of tc is important .
our case was consistent with the mayo criteria for the clinical diagnosis of tc . in summary | takotsubo cardiomyopathy ( tc ) is a rare reversible circulatory syndrome often detected in elderly patients after general surgery .
we report the case of an 85-year - old woman who was admitted for obstructive jaundice and underwent ercp and enbd before operation .
she finally underwent cholecystectomy and choledocholithotomy with the diagnosis of gallbladder and common bile duct stones . in the evening
just after the operation , without symptoms of cardiac failure , her ecg suddenly exhibited abnormal st - t elevation in leads ii , iii , and v2-v6 , though neither cpk(mb ) nor troponin t was elevated .
echocardiogram demonstrated basal hyperkinesis and apical dyskinesis , suggesting tc .
conservative treatment enabled recovery , ecg was normalized in a month , and the postoperative course was satisfactory .
the stress on her during treatment might have been a cause of this unique cardiomyopathy , and it should be recalled that tc is a potential complication of hepato - biliary - pancreatic surgery . |
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | the international conference on structural genomics ( icsg 2011 , http://www.icsg2011.org ) , held in toronto canada may 1014 , 2011 was a rich and exciting demonstration of how far structural genomics has come .
structural genomics has now matured into a field that includes both structure and the biology that structure enables .
this has allowed targeting based on systematic approaches and on known biological importance and allows biochemical studies to be closely tied to structure determination .
the wealth of purified proteins , clones , and chemical probes produced by structural genomics groups will enable a vast amount of follow - on research .
the technologies , the structures , and the biology that were described at the meeting were at the cutting edge of science .
structural genomics has become a success . |
despite renewed interest in malaria control and eradication , malaria remains a worldwide burden , with nearly half of the world s population at risk of infection and more than 550,000 deaths annually , particularly children in sub - saharan africa .
these new control and eradication programs have been supported by the genomic renaissance , with large numbers of malaria parasites sequenced and analyzed for mutations associated with reduced drug sensitivity , increased virulence , and for population characteristics .
single - nucleotide polymorphisms ( snps ) are among the most commonly identified genetic variants .
portable snp genotyping methods offer on - site and real - time population surveillance and tracking .
in addition to fingerprinting individual parasites , the molecular barcode is also used to detect dramatic temporal shifts in allele frequency as well as variance effective population size and complexity of infection .
while this set of informative snps is easily adapted to many genotyping platforms , high resolution melting ( hrm ) analysis is particularly well - suited to field - based studies , where sensitive and simple operation and detection of novel mutations at low costs compared to sequencing and other approaches are attractive in resource - poor settings .
hrm starts with standard polymerase chain reaction ( pcr ) that incorporates a fluorescent dye .
post - pcr melting analysis determines the peak amplicon melting temperature ; a single snp difference in a short amplicon can result in substantial peak melting temperature ( tm ) differences .
several refinements to this method offer better genotyping resolution to differentiate snps including class iv ( a - t ) snps , and detect minor mutant alleles in samples with multiple alleles present ( polygenomic infections ) .
first , the assays incorporate short probes centered over the snp region in addition to forward and reverse primers that are present at different concentrations .
these blocked probes are not amplified during pcr , but bind to the strand produced in excess due to asymmetric primer concentrations that increase production of the probe - template product .
these separate double - stranded amplicons consisting of probe bound to the excess template strand are ~20 - 30 bp ; their significantly decreased length compared to the entire amplicon ( 80 - 150 bp ) lead to much larger tm differences associated with single or multiple base probe - template mismatches .
second , mutant allele amplification bias ( maab ) lowers the reaction annealing temperature to bias the reaction towards mutant alleles present at low ratios in polygenomic infections .
the annealing temperature is set between the tms of perfectly matched ( wild - type ) and mismatched ( mutant ) probes .
at this temperature , the binding of the wild - type allele is stable enough that amplification is hindered compared to its mismatch equivalent , thus biasing the amplification towards the mutant allele when both are present in a single sample . with these hrm refinements ,
this technology has allowed tracking of origins and identity of parasites associated with epidemic infections in south america and detection of new mutations , differentiation of multiple snps located immediately next to each other in sequence , and mutations present in less than 1% of the alleles in polygenomic samples .
increased sensitivity is particularly important in regions approaching pre - elimination status and those at risk for emerging drug resistance .
the ability to quickly and easily identify imported parasites and snps associated with reduced sensitivity on - site informs surveillance efforts and control programs about the effectiveness of their implementations and identifies hotspots for increased malaria eradication and elimination efforts .
this protocol describes the methods for blocked - probe - based and maab for increased hrm genotyping sensitivity .
note : this protocol includes volumes and concentrations for 96-well plate , 8-tube strips , and capillary - based systems ( 10 l reaction volume ) ; however , hrm can also be performed in 384-well plate - based systems with a 5 l reaction volume by scaling all volumes correspondingly .
the range of detection for most systems is 10 pg to 10 ng template dna .
obtain plasmodium falciparum samples directly from blood obtained from patients participating in field site studies and stored as whole blood , pelleted red blood cells , or on filter paper .
note : samples may also be culture - adapted to grow in vitro ; some standard laboratory strains for analysis can be ordered from the malaria research and reference reagent resource center ( mr4 , samples may be used directly from pelleted red blood cells or culture or extracted from whole blood , red blood cells , or filter paper .
dna extracted from filter paper or culture - adapted strains note : hrm is sensitive to salt concentration ; concentration differences due to variable extraction methods can significantly affect melting temperatures . while specific final buffers are not a significant factor for success , use a standard common elution or resuspension buffer ( i.e. , water , 1x te [ low te or dna suspension buffer : 10 mm tris - cl , 0.10 mm edta ] or 1x te [ 10 mm tris - cl , 1 mm edta ] ) for all samples to avoid anomalous or inconsistent melt curves . prepare template dilutions of 10 pg 10 ng per l in standard te , te , or water for each 10 l reaction .
prepare template dilutions of 10 pg 10 ng per l in standard te , te , or water for each 10 l reaction . direct amplification from pelleted red blood cells determine parasitemia of red blood cells using thin - smear microscopy note : methods for determining parasitemia of infected red blood cells are available from the centers for disease control and prevention ( cdc ) : http://www.cdc.gov/dpdx/diagnosticprocedures/blood/microexam.htmldilute parasitemias above 0.5% 1:400 in pcr - grade water , te , or te . use 3 l for each 5 10 l reaction .
determine parasitemia of red blood cells using thin - smear microscopy note : methods for determining parasitemia of infected red blood cells are available from the centers for disease control and prevention ( cdc ) : http://www.cdc.gov/dpdx/diagnosticprocedures/blood/microexam.html dilute parasitemias above 0.5% 1:400 in pcr - grade water , te , or te .
controls note : hrm can be used for gene scanning to detect sequence variants in a population ; however , accurate genotyping requires standards sequence - verified to contain the snp being interrogated .
plasmid constructs or malaria isolates with known genotypes can be used . for most applications , 3d7 ( mr4 mra-151 )
is used as a wild - type control . due to between - run variability , always include positive and negative controls .
positive controls : prepare 10 pg 10 ng per 1 l dilutions of plasmid or standards with sequence - verified snp genotypes.negative control : use pcr grade water as a no - template control ( ntc ) for the amplification reactions .
positive controls :
prepare 10 pg 10 ng per 1 l dilutions of plasmid or standards with sequence - verified snp genotypes .
negative control : use pcr grade water as a no - template control ( ntc ) for the amplification reactions .
note : some hrm systems are standalone melting platforms and do not have a heated lid to prevent condensation and product evaporation during amplification .
add 20 l light mineral oil to each well plate before loading the reaction mixture .
prepare 10x working stocks of all assays refer to table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked - probe - based hrm genotyping .
prepare reaction mixtures to each well in plate or tube , add 1 l 10x working stock forward and reverse primers and probes , 4.0 5.0 l 2.5x or 2.0x hrm master mix , 1 l template , and pcr grade water for a total reaction volume of 10 l .
cover plates or tubes use optical plate seals for plate - based amplification , optical caps for strip - tube - based systems , and plastic caps for capillary - based systems .
be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification .
spin samples spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used .
note : some hrm systems are standalone melting platforms and do not have a heated lid to prevent condensation and product evaporation during amplification .
add 20 l light mineral oil to each well plate before loading the reaction mixture .
add 20 l light mineral oil to each well plate before loading the reaction mixture .
prepare 10x working stocks of all assays refer to table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked - probe - based hrm genotyping .
refer to table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked - probe - based hrm genotyping .
prepare reaction mixtures to each well in plate or tube , add 1 l 10x working stock forward and reverse primers and probes , 4.0 5.0 l 2.5x or 2.0x hrm master mix , 1 l template , and pcr grade water for a total reaction volume of 10 l .
to each well in plate or tube , add 1 l 10x working stock forward and reverse primers and probes , 4.0 5.0 l 2.5x or 2.0x hrm master mix , 1 l template , and pcr grade water for a total reaction volume of 10 l .
cover plates or tubes use optical plate seals for plate - based amplification , optical caps for strip - tube - based systems , and plastic caps for capillary - based systems .
be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification .
use optical plate seals for plate - based amplification , optical caps for strip - tube - based systems , and plastic caps for capillary - based systems .
be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification .
spin samples spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used .
spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used .
run standard blocked - probe or maab amplification protocols ( tables 2 and 3 , respectively ) .
run standard blocked - probe or maab amplification protocols ( tables 2 and 3 , respectively ) .
note the difference in annealing temperature between these methods . melt after pcr amplification , proceed to melting analysis . for standalone melting systems that do not have built - in amplification ( e.g. , biofire defense lightscanner systems ) , remove amplified plate from thermal cycler and place in hrm instrument . from the system software interface ,
note : to save melting and analysis time , the melting window can be shortened to cover temperature ranges for just probe or amplicon melting regions .
after melting , plates and tubes can be re - melted if necessary and stored at -20 c or 4 c .
for standalone melting systems that do not have built - in amplification ( e.g. , biofire defense lightscanner systems ) , remove amplified plate from thermal cycler and place in hrm instrument . from the system software interface ,
note : to save melting and analysis time , the melting window can be shortened to cover temperature ranges for just probe or amplicon melting regions .
after melting , plates and tubes can be re - melted if necessary and stored at -20 c or 4 c .
melt analysis remove negative samples within the analysis software , remove from further analysis samples that did not amplify or that have jagged melting peaks .
note : after selecting the mode of analysis for an individual run file , the instrument automatically groups the positive and negative samples . before genotyping each subset , the user can manually alter the calls . from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click
normalize from the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) ( difference curves ) view , move normalization bars to surround the probe melt region .
note : the probe melt region is the lower temperature of the two melting regions .
calculate groups after normalization , select calculate groups to automatically assign samples to groups .
note : some analysis software has high sensitivity thresholds that can not be changed ; in these cases , the software might assign samples to different groups that visually belong to another .
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping
tab.enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press ok and save the results .
remove negative samples within the analysis software , remove from further analysis samples that did not amplify or that have jagged melting peaks .
note : after selecting the mode of analysis for an individual run file , the instrument automatically groups the positive and negative samples . before genotyping each subset , the user can manually alter the calls . from either the melting curve or melting
peak view , right - click on the curves to be removed to select them . after selecting the misclassified samples ,
go to new call , select the appropriate grouping , and click apply change .
within the analysis software ,
remove from further analysis samples that did not amplify or that have jagged melting peaks .
note : after selecting the mode of analysis for an individual run file , the instrument automatically groups the positive and negative samples . before genotyping each subset , the user can manually alter the calls . from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click apply change .
from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click apply change .
normalize from the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) ( difference curves ) view , move normalization bars to surround the probe melt region . note : the probe melt region is the lower temperature of the two melting regions .
the normalization bars should be at the base of the melt peaks .
from the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) ( difference curves ) view , move normalization bars to surround the probe melt region .
note : the probe melt region is the lower temperature of the two melting regions .
calculate groups after normalization , select calculate groups to automatically assign samples to groups .
note : some analysis software has high sensitivity thresholds that can not be changed ; in these cases , the software might assign samples to different groups that visually belong to another .
after normalization ,
select calculate groups to automatically assign samples to groups . if necessary , manually re - assign samples to specific groups .
note : some analysis software has high sensitivity thresholds that can not be changed ; in these cases , the software might assign samples to different groups that visually belong to another .
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping
tab.enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping
tab.enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press ok and save the results .
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping tab . enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press
obtain plasmodium falciparum samples directly from blood obtained from patients participating in field site studies and stored as whole blood , pelleted red blood cells , or on filter paper .
note : samples may also be culture - adapted to grow in vitro ; some standard laboratory strains for analysis can be ordered from the malaria research and reference reagent resource center ( mr4 , samples may be used directly from pelleted red blood cells or culture or extracted from whole blood , red blood cells , or filter paper .
dna extracted from filter paper or culture - adapted strains note : hrm is sensitive to salt concentration ; concentration differences due to variable extraction methods can significantly affect melting temperatures . while specific final buffers are not a significant factor for success , use a standard common elution or resuspension buffer ( i.e. , water , 1x te [ low te or dna suspension buffer : 10 mm tris - cl , 0.10 mm edta ] or 1x te [ 10 mm tris - cl , 1 mm edta ] ) for all samples to avoid anomalous or inconsistent melt curves .
prepare template dilutions of 10 pg 10 ng per l in standard te , te , or water for each 10 l reaction .
prepare template dilutions of 10 pg 10 ng per l in standard te , te , or water for each 10 l reaction . direct amplification from pelleted red blood cells determine parasitemia of red blood cells using thin - smear microscopy note : methods for determining parasitemia of infected red blood cells are available from the centers for disease control and prevention ( cdc ) : http://www.cdc.gov/dpdx/diagnosticprocedures/blood/microexam.htmldilute parasitemias above 0.5% 1:400 in pcr - grade water , te , or te .
determine parasitemia of red blood cells using thin - smear microscopy note : methods for determining parasitemia of infected red blood cells are available from the centers for disease control and prevention ( cdc ) : http://www.cdc.gov/dpdx/diagnosticprocedures/blood/microexam.html dilute parasitemias above 0.5% 1:400 in pcr - grade water , te , or te .
controls note : hrm can be used for gene scanning to detect sequence variants in a population ; however , accurate genotyping requires standards sequence - verified to contain the snp being interrogated .
plasmid constructs or malaria isolates with known genotypes can be used . for most applications , 3d7 ( mr4 mra-151 )
is used as a wild - type control . due to between - run variability , always include positive and negative controls .
positive controls : prepare 10 pg 10 ng per 1 l dilutions of plasmid or standards with sequence - verified snp genotypes.negative control : use pcr grade water as a no - template control ( ntc ) for the amplification reactions .
positive controls : prepare 10 pg 10 ng per 1 l dilutions of plasmid or standards with sequence - verified snp genotypes .
negative control : use pcr grade water as a no - template control ( ntc ) for the amplification reactions .
prepare reaction mixture ( optional ) mineral oil overlay . note : some hrm systems are standalone melting platforms and do not have a heated lid to prevent condensation and product evaporation during amplification .
add 20 l light mineral oil to each well plate before loading the reaction mixture .
prepare 10x working stocks of all assays refer to table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked - probe - based hrm genotyping .
prepare reaction mixtures to each well in plate or tube , add 1 l 10x working stock forward and reverse primers and probes , 4.0 5.0 l 2.5x or 2.0x hrm master mix , 1 l template , and pcr grade water for a total reaction volume of 10 l .
cover plates or tubes use optical plate seals for plate - based amplification , optical caps for strip - tube - based systems , and plastic caps for capillary - based systems .
be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification .
spin samples spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used .
note : some hrm systems are standalone melting platforms and do not have a heated lid to prevent condensation and product evaporation during amplification .
add 20 l light mineral oil to each well plate before loading the reaction mixture .
add 20 l light mineral oil to each well plate before loading the reaction mixture .
prepare 10x working stocks of all assays refer to table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked - probe - based hrm genotyping .
refer to table 1 for recommended 10x working stock concentrations as well as final concentrations for blocked - probe - based hrm genotyping .
prepare reaction mixtures to each well in plate or tube , add 1 l 10x working stock forward and reverse primers and probes , 4.0 5.0 l 2.5x or 2.0x hrm master mix , 1 l template , and pcr grade water for a total reaction volume of 10 l .
to each well in plate or tube , add 1 l 10x working stock forward and reverse primers and probes , 4.0 5.0 l 2.5x or 2.0x hrm master mix , 1 l template , and pcr grade water for a total reaction volume of 10 l .
cover plates or tubes use optical plate seals for plate - based amplification , optical caps for strip - tube - based systems , and plastic caps for capillary - based systems .
be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification .
use optical plate seals for plate - based amplification , optical caps for strip - tube - based systems , and plastic caps for capillary - based systems .
be sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation during amplification .
spin samples spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used .
spin plates 3 min at 1,800 x g to remove air bubbles and to separate oil and aqueous phases if mineral oil is used . amplification protocol place reaction plates or tubes in thermal cycler .
run standard blocked - probe or maab amplification protocols ( tables 2 and 3 , respectively ) .
run standard blocked - probe or maab amplification protocols ( tables 2 and 3 , respectively ) .
melt after pcr amplification , proceed to melting analysis . for standalone melting systems that do not have built - in amplification ( e.g. , biofire defense lightscanner systems ) ,
remove amplified plate from thermal cycler and place in hrm instrument . from the system software interface ,
note : to save melting and analysis time , the melting window can be shortened to cover temperature ranges for just probe or amplicon melting regions .
after melting , plates and tubes can be re - melted if necessary and stored at -20 c or 4 c .
after pcr amplification , proceed to melting analysis . for standalone melting systems that do not have built - in amplification ( e.g. , biofire defense lightscanner systems ) , remove amplified plate from thermal cycler and place in hrm instrument . from the system software interface , melt the plate from 40
note : to save melting and analysis time , the melting window can be shortened to cover temperature ranges for just probe or amplicon melting regions .
after melting , plates and tubes can be re - melted if necessary and stored at -20 c or 4 c .
melt analysis remove negative samples within the analysis software , remove from further analysis samples that did not amplify or that have jagged melting peaks .
note : after selecting the mode of analysis for an individual run file , the instrument automatically groups the positive and negative samples . before genotyping each subset , the user can manually alter the calls . from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click apply change .
normalize from the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) ( difference curves ) view , move normalization bars to surround the probe melt region .
note : the probe melt region is the lower temperature of the two melting regions .
calculate groups after normalization , select calculate groups to automatically assign samples to groups .
note : some analysis software has high sensitivity thresholds that can not be changed ; in these cases , the software might assign samples to different groups that visually belong to another .
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping
tab.enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press ok and save the results .
remove negative samples within the analysis software , remove from further analysis samples that did not amplify or that have jagged melting peaks .
note : after selecting the mode of analysis for an individual run file , the instrument automatically groups the positive and negative samples . before genotyping each subset , the user can manually alter the calls . from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click apply change .
within the analysis software ,
remove from further analysis samples that did not amplify or that have jagged melting peaks .
note : after selecting the mode of analysis for an individual run file , the instrument automatically groups the positive and negative samples . before genotyping each subset , the user can manually alter the calls . from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click apply change .
from either the melting curve or melting peak view , right - click on the curves to be removed to select them .
after selecting the misclassified samples , go to new call , select the appropriate grouping , and click apply change .
normalize from the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) ( difference curves ) view , move normalization bars to surround the probe melt region . note : the probe melt region is the lower temperature of the two melting regions .
the normalization bars should be at the base of the melt peaks .
from the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) ( difference curves ) view , move normalization bars to surround the probe melt region .
note : the probe melt region is the lower temperature of the two melting regions .
calculate groups after normalization , select calculate groups to automatically assign samples to groups .
note : some analysis software has high sensitivity thresholds that can not be changed ; in these cases , the software might assign samples to different groups that visually belong to another .
after normalization ,
select calculate groups to automatically assign samples to groups . if necessary , manually re - assign samples to specific groups .
note : some analysis software has high sensitivity thresholds that can not be changed ; in these cases , the software might assign samples to different groups that visually belong to another . assign genotypes
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping
tab.enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press ok and save the results .
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping
tab.enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press
after the user confirms that the computed groups are correct ( if not , that correct changes have been made under the grouping tab by selecting misclassified samples and selecting the right group from the drop - box beside new call ) , select edit group names under the grouping tab . enter the genotypes of the standards into the corresponding colored box ( for example , if standard 3d7 has a known a genotype and is in the red group , the red group has genotype a ) . press ok and save the results .
data can be visualized in several different ways , depending on the analysis software and instrument .
typically , a plot of the negative derivative of normalized fluorescence with respect to temperature ( df / dt ) against temperature results is most straightforward for visualizing melting peaks and determining genotypes . a full melting window ( 40c80c )
figure 1 shows an example of normalized melting peaks for both regions . setting the analysis window to just the probe region results in clear differentiation of peaks corresponding to specific snps ( figure 2 ) .
probe - based analysis clearly shows the presence of both alleles as individual peaks with melting temperatures that match homozygous snp peaks ( figure 3 ) .
figure 4 shows that progressively reducing the annealing temperature during amplification ( maab ) results in a bias towards the mutant allele ( left - side peak ) ( figure 4a ) , resulting in increased sensitivity for the mutant allele in a population of mixed alleles ( figure 4b ) .
figure 1 . probe and amplicon melting regions . plotting the negative derivative of the normalized fluorescence with respect to temperature over a wide melting window results in both probe ( lower temperature ) and amplicon melting peaks that can be analyzed .
doi : 10.1128/aac.05737 - 11 ) please click here to view a larger version of this figure .
perfect matches ( typically wild - type alleles ) result in higher melting peaks ( red ) , while snp mismatches have lower melting temperatures ( grey ) .
when both alleles are present in a sample , probe - based hrm analysis represents both alleles as two - peak curves ( orange ) with peaks that match single - allele samples ( red and grey ) .
doi : 10.1128/aac.05737 - 11 ) please click here to view a larger version of this figure .
a. progressively lowering the amplification annealing temperature biases the melting peak reaction towards peak corresponding to the mutant ( lower temperature ) allele in a polygenomic or polyallelic sample .
b. maab results in hrm sensitivity to detect minor alleles present at less than 1% in polygenomic or polyallelic samples .
doi : 10.1128/aac.05737 - 11 ) please click here to view a larger version of this figure .
table 1 .
set - up of probe - based high resolution melting amplification reactions .
continous hrm is a post - pcr analysis step ; therefore , sample and assay setup is similar to standard pcr protocols , with the additional incorporation of a fluorescent intercalating dye used to track the transition from double - stranded to single - stranded dna during the high - resolution melting step .
assay design is key , and several tools optimized for hrm assay design are available commercially and online .
amplicon lengths of 80 - 150 base pairs with accompanying ~20 base pair blocked probes centered over the snp or snps of interest work best to differentiate snps as well as haplotypes of multiple snps within the probe region .
probes can be blocked using either a 3 c3 spacer or a 2 base pair mismatch at the 3 end , which prevent extension during amplification .
probes can be designed to match the watson or crick template strand ; the choice depends on empirical testing to determine which probe design produces acceptable melting peaks .
excess forward or reverse primers are used to produce single - stranded amplification product that anneals to the blocked probes .
if the probe contains the forward strand sequence , then reverse primer is used in excess , typically in a 1:5 ratio , and vice - versa for probes that match the reverse strand .
pcr reaction optimization requires gradient pcr and agarose gels or bioanalyzer analysis to determine optimal annealing temperatures .
template concentration can be increased using methods such as pre - amplification , biased multiplexed amplification of all targets using low primer concentration and cycle numbers to increase the template concentration .
the thermal properties of the glass capillaries tubes used in these systems facilitate this method .
the small interior diameter of the capillaries as well as the rapid heat transfer through glass offer more stringent temperature control than standard pcr plasticware , which has slower transition rates between annealing and denaturation cycles due to the thermal insulating properties of the plastics . with this method ,
detection sensitivities can be reduced from 2 - 5% to less than 1% of a mutant allele in a mixture of wild - type and mutant alleles .
hrm is a facile , efficient , and economical tool for snp genotyping in a variety of settings and numerous applications , from infectious disease surveillance to scanning for cancer gene variants .
several other instruments , such as the roche nano and lightcycler systems ( 480 and 96 ) as well as the eco real - time pcr system offer hrm in addition to standard amplification , real - time , and copy - number functionality . using higher - throughput machines , hrm barcoding costs less than $ 0.50 per assay in our hands , including all reagents and consumables . in the context described here , field - based applications allow real - time population surveillance for changes associated with malaria control efforts , including reduced population diversity , detection of imported parasite types , and the appearance and spread of snps associated with reduced drug sensitivity
. refinements to the method offer additional sensitivity useful for informing progress towards malaria elimination and eradication .
| despite decades of eradication efforts , malaria remains a global burden .
recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about plasmodium parasite species responsible for malaria , including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti - malarial drugs.one common genetic variation , single - nucleotide polymorphisms ( snps ) , offers attractive targets for parasite genotyping .
these markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.snp genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance , particularly in response to malaria control efforts in regions nearing elimination status.while informative snps have been identified that are agnostic to specific genotyping technologies , high - resolution melting ( hrm ) analysis is particularly suited to field - based studies .
compared to standard fluorescent - probe based methods that require individual snps in a single labeled probe and offer at best 10% sensitivity to detect snps in samples that contain multiple genomes ( polygenomic ) , hrm offers 2 - 5% sensitivity .
modifications to hrm , such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias pcr towards amplification of the minor allele , further increase the sensitivity of hrm . while the sensitivity improvement depends on the specific assay , we have increased detection sensitivities to less than 1% of the minor allele.in regions approaching malaria eradication , early detection of emerging or imported drug resistance is essential for prompt response . similarly , the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.this manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples . |
hydatid disease had been recognized since the galen and hipocrates , and in 1808 rudolphi used the term hydatid cyst to describe echinococcosis in humans .
the most common location of a hydatid cyst is liver and lungs at the second place ( 10 - 40% ) .
generally , a pulmonary hydatid cyst is diagnosed by radiological imaging , physical examination and history findings .
the surgical procedures may be conservative ( cystostomy , enucleation of intact cysts , removal of the cyst after needle aspiration with or without pericystectomy , with maximal preservation of lung parenchyma ) or radical ( lung parenchyma resection with cystotomy and pericystectomy , wedge resection , segmentectomy and lobectomy ) .
however , the selection of surgical technique depends on the conditions throughout surgery . while hydatid disease is endemic in iran and the highest rate
is reported for khorasan province and few studies have evaluated the relation between location of the cysts and type of surgical technique , we aimed to perform this study to assess surgical treatment of pulmonary hydatid cysts and evaluate whether the location of a cyst affects the choice of surgical technique .
in this study , the patients with a pulmonary hydatid cyst who had been referred to thorax surgery section of qaem hospital related to mashhad university of medical sciences from 2010 to 2012 were evaluated . among 99 patients ,
12 cases were excluded because 6 patients had multiple bilateral lung hydatid cysts and they treated medically , 4 patients were inoperable due to other medical problem and 2 patients rejected surgical treatment .
initially , all conditions of the study were completely described for the patients and in case of their assent ; they would sign a written informed consent .
moreover , the study was approved by the regional ethics committee of mashhad university of medical sciences ( project number : 900415 ) .
the studied variables included age , gender , clinical symptoms , type of surgery , postoperative complications , number of hydatid cysts in lungs and their size and location , hospitalization , bilateral lung hydatid cyst and mortality .
diagnosis was definite in the patients referred as elective . for the patients who had been referred as emergency , after obtaining history and physical examination , chest x - ray and ct scan were performed . for the cases with unsure diagnosis ,
the surgical technique approach was based on the location , size and number of cysts .
preoperative evaluation was performed . among 87 patients , for 14 cases with over 40 years old we did an electrocardiography and in 13 patients with history of chronic lung disease ,
lung function tests were performed . according to the results of these tests and having no contraindication for surgery ,
all the cases were put in decubitus lateral position and posterolateral thoracotomy was performed through the fifth , sixth or seventh ( depending on the location of the cyst ) intercostal spaces . to prevent contamination of thoracotomy site and the surrounding tissue with hydatid cyst fluid ,
the surrounding of cyst was trapped with gas containing hypertonic sodium chloride solution 20% . during the operation ,
two suctions were ready ; one inserted into trocar and the other in terminal suction . according to the location , number and size of cysts ,
we selected each of the following operation techniques : cystotomy and bronchial closure and capitonnage , cystotomy and bronchial closure , segmentectomy , enucleation , and lobectomy . then , the patients were evaluated for the relation between the location of a cyst and the choice of surgery .
the patients were followed up for 1 month in terms of postoperative complications . to describe data , statistical charts and tables were used and data analysis was performed by spss version 19 .
the chi - square test was used to compare treatment outcome and morbidity in the different methods of surgery and variance analysis was used to compare the duration of hospitalization in the different methods of surgery .
before the operation , the anesthetist was asked to start corticosteroids to prevent anaphylactic shock .
all the cases were put in decubitus lateral position and posterolateral thoracotomy was performed through the fifth , sixth or seventh ( depending on the location of the cyst ) intercostal spaces . to prevent contamination of thoracotomy site and the surrounding tissue with hydatid cyst fluid ,
the surrounding of cyst was trapped with gas containing hypertonic sodium chloride solution 20% . during the operation ,
two suctions were ready ; one inserted into trocar and the other in terminal suction . according to the location , number and size of cysts , we selected each of the following operation techniques : cystotomy and bronchial closure and capitonnage , cystotomy and bronchial closure , segmentectomy , enucleation , and lobectomy
. then , the patients were evaluated for the relation between the location of a cyst and the choice of surgery .
the patients were followed up for 1 month in terms of postoperative complications . to describe data , statistical charts and tables were used and data analysis was performed by spss version 19 .
the chi - square test was used to compare treatment outcome and morbidity in the different methods of surgery and variance analysis was used to compare the duration of hospitalization in the different methods of surgery .
a total of 87 patients with diagnosis of a pulmonary hydatid cyst were evaluated . among them , 46 cases ( 52.9% ) were males and 41 ( 47.1% ) were females .
the most affected age group was patients in their third decade with a mean age of 29.61 14.27 ( minimum 7 years and maximum 70 years )
symptoms of a pulmonary hydatid cyst in the studied groups the incidence of hepatic involvement was 19.5% ( 17 cases ) and the hepatic localization was not complicated in the chest in16 patients .
we operated a lung hydatid cyst at first and then a liver cyst operated in the second stage ( 6 weeks later ) .
one patient was referred due to biloptysis and he was treated for both lung and liver cysts in first stage .
we found a cyst in the middle lobe and fistula between diaphragm and lower lobe .
then diaphragm was opened and the hepatic cyst was drained and was capitonized . sub - diaphragmatic drain and chest tube
the rate of bilateral hydatid cyst was 22 cases ( 25.3% ) and unilateral hydatid cyst was 65 cases ( 74.7% ) . among 87 patients , 61 cases ( 70.1% ) had a cyst with size of < 10 cm and 26 ( 29.9% ) had cyst with size of 10 cm . in terms of the number of cysts , 5 patients ( 5.7% ) had more than three cysts and 67 cases ( 77% ) one cyst , 9 ( 10.3% ) two cysts , and 6 ( 6.9% ) three cysts .
about the location of the cysts , in 19 cases ( 21.8% ) , the location of cyst was right lower lobe , in 12 ( 13.8% ) right upper lobe , in 26 ( 29.9% ) left lower lobe , in 11 ( 12.6% ) left upper lobe , in 3 ( 3.4% ) middle lobe , in 5 ( 5.7% ) we observed pleural involvement and multi - lobe involvement in 11 ( 12.6% ) .
the surgical technique approach according to the location of the cysts in the patients with a pulmonary hydatid cyst is shown in table 2 .
there was significant relation between the existence of the cyst in the middle lobe and performing lobectomy ( p = 0.016 ) .
type of operation according to the location of cysts the choice of operation technique according to the size of the cysts in the patients with a pulmonary hydatid cyst is shown in table 3 .
no significant relation was found between the size of cyst and lobectomy ( p = 0.682 ) .
type of operation according to the size of cysts selection of operation technique according to the number of the cysts in the patients with pulmonary hydatid cyst is shown in table 4 .
no significant relation was observed between the number of cysts and lobectomy ( p = 0.344 ) .
the results show that the choice of surgical technique is based on the location , not the size and number of cysts .
type of operation according to the number of cysts in terms of hospitalization , nine cases had hospital stay for more than 9 days and five of them had undergone lobectomy .
the most common postoperative complication in the studied patients was prolonged air leak , which occurred in 12 cases .
other postoperative complications were empyema in 1 case , hemoptysis in 5 patients and bile secretion from drain in 1 case . in the last patient we performed conservative treatment because the patient was severely ill .
we managed bleeding process during operation but the patient expired 2 days later in icu due to multiorgan failure .
96.8% had no severe problem and 3.2% had complication and 25 patients did nt refer for follow - up .
it is an important health problem in regions where people earn the living by agriculture and livestock raising , while veterinary services , public health and preventive policies are poorly offered .
common complaints of patients with lung hydatid cysts are dry cough , hemoptysis and feeling pressure in the thorax .
hydatid disease when is located in lungs can reach a certain size without causing any symptom .
a cyst is identifiable in chest x - ray and computed tomography ( ct ) scanning as a round or oval homogenous opacity that can be differentiated from pulmonary parenchyma .
some studies reported that in patients with pulmonary hydatid disease , the lung parenchyma should be preserved as much as possible and radical procedures must be avoided .
reported that surgical treatment is related to size , localization , clinical manifestation and eventual complication of the cysts .
the most common localization of a hydatid cyst is the liver ( 50 - 60% ) and secondly the lungs ( 10 - 30% ) .
a hydatid cyst should be treated as soon as diagnosis is made , since it may cause serious complications by means of rupture into bronchi and pleural cavity or vital organ compression .
medical treatment is considered for small cysts without complication , in patients who are high risk for surgery and those who reject the surgery .
the choice of surgical technique is based on the location , size and the number of cysts . since
, hydatid disease is endemic in some parts of the world including iran , we aimed to perform this study to evaluate whether the location of cyst affects the choice of surgical technique or not . in this study ,
the most affected age group was 21 - 30 years with a mean age of 30 years and when compared with other studies , the same age group was involved . although it may occur in any age group , it is more common in the third and fourth decades and is more prevalent in men . in this study ,
the rate of incidence of a pulmonary hydatid cyst was 52.9% in male and 48.1% in female . in a study by bulent et al .
no significant differences were found between the incidence rates by the gender . however , our result about the gender - related incidence rate was similar to those reported in the study performed by bilgin et al .
but , the study of ghaffarifar reported that the incidence rate is more common in females . in the referred patients of this study ,
this was similar to the study of bagheri et al . which reported that in the patients with a pulmonary hydatid cyst , cough and chest pain were the most common complaints ( 62% , 56% , respectively ) .
62 males and 40 females , reported that there is a relation between symptoms of patients and localization and size of the hydatid lung cysts . in this study ,
the incidence of hepatic involvement was 19.5% and the rate of bilateral hydatid cyst was 25.3% .
a series of 1055 patients reported that the incidence rate of concomitant hepatic and pulmonary involvement was less than 10% . in the study of karavdic
, 70.1% of the patients had a cyst with size of < 10 cm and 29.9% had a cyst with size of 10 cm .
no significant relation was found between the size of cyst and lobectomy ( p = 0.682 ) .
usluer et al . performed a study on two groups of the patients with a hydatid cyst : those smaller than 10 cm ( group a ) and those 10 cm or greater ( group b ) .
there were 462 patients ( 86% ) in group a and 75 patients ( 14% ) in group b. lobectomy rates were significantly higher in group b compared with group a ( p = 0.038 ) . also , isitmangil et al . reported that one of the indications for lobectomy is large cysts involving more than 50% of the lobe
moreover , in this study , we did nt find any significant relation between the number of cysts and performing lobectomy ( p = 0.344 ) .
however , the literature review demonstrated one of the major indications of lobectomy , the number and not the size of the cysts . in our study , the left lower lobe was the most frequently affected area of the lung ( 29.9% ) and the right lower lobe was affected as 21.8% .
concluded that the right lower lobe was the most frequently attacked area of the lung .
the study of sehitogullari showed that the right lower lobe was the most affected area of the lung .
also , in the present study , there was a significant relation between the existence of a cyst in the middle lobe and lobectomy ( p = 0.016 ) .
stated that lobectomy should be performed in cases where more than half of the lobe is involved .
of course , no study has been performed so far to evaluate the relation between the location of cysts and lobectomy .
postoperative morbidity rates have varied between 0% to 17% , with air leak as the most common complication . in our study also , the most common postoperative complication was air leak in 12 patients of which 25% of cases of air leak was observed in the patients who underwent lobectomy .
there was a significant relation between postoperative air leak and lobectomy ( p = 0.052 ) . in the medical literature ,
mortality rates ranged from 0% to 2% . in our study , the rate of mortality was 1.15% .
in spite of having not enough cases ( 89 patients ) and cases with a middle lobe cyst were few ( 7 patients ) , our data indicate if a hydatid cyst is located in the middle lobe , it may need lobectomy .
although size and number of cyst are determining factors for the selection of operation , but the location and the ratio of destruction of lung are important factors too . also , in patients with underlying pulmonary diseases if the lesion is completely removable with lobectomy , it is better not to insist on conservative surgical therapy , because it leads to incomplete tissue removal and therefore more complications would be expected .
moreover , since , pulmonary hydatid disease is endemic in the area of khorasan , registry of the cases for better organizing the health , medical and statistical burden and prevalence of this disease is recommended .
however , more studies with larger sample size are required to obtain more accurate results . | purpose : a hydatid cyst is the most common lung parasitic disease and is endemic in iran .
a hydatid cyst is more common in the right lung and lower lobes.objective:the aim of this study was to assess surgical treatment of pulmonary hydatid cysts and whether the location of cyst affects surgical technique approaches.materials and methods : this study was performed on 87 patients with a pulmonary hydatid cyst who were referred to qaem hospital from 2010 to 2012 .
selection of surgical technique was according to size , location , and the number of cysts .
patients were divided into two groups : ( 1 ) surgery with preserving lung parenchyma and ( 2 ) lobectomy .
afterward , the relationship between the location of cyst and surgical technique approaches was evaluated .
data was analyzed by chi - square and fisher exact tests.results:in this study , no significant relation was found between the size ( p = 0.682 ) and number ( p = 0.344 ) of cysts and lobectomy necessity .
but , there was a significant relation between the occurrence of cyst in middle lobe and lobectomy necessity ( p = 0.016).conclusions : according to the results , type of surgical technique does not depend on the size and number of cysts , but it may be related to the location of the cyst and the ratio of lung destruction . |
periodontitis is a set of chronic inflammatory disorders of the tissues surrounding the teeth leading to the destruction of the alveolar bone and teeth loss .
periodontitis caused an increase in the proliferation of human periodontal ligament stem cells and a decrease in the commitment to the osteoblast lineage .
endothelin-1 is the most potent and predominant endothelin isoform in the human cardiovascular system and is now recognized to play a pivotal role in the regulation of vascular tone .
et-1 stimulates neutrophils to release elastase , activates mast cells , and stimulates monocytes to produce a variety of cytokines such as interleukin- ( il- ) 1 , il-6 , and tumor necrosis factor- ( tnf- ) , which play important roles in the initiation and progression of periodontal disease .
it has recently been found that porphyromonas gingivalis , one of the major pathogens responsible for causing periodontitis , stimulates the expression of et-1 , with upregulation of proinflammatory cytokines and intercellular adhesion molecule , in the bronchial epithelial cell line hep-2 and kb cells .
the expression of et-1 was strongly enhanced in gingival tissue and endothelial cells during periodontal inflammation .
the periodontal ligament ( pdl ) is a special connective tissue located between the alveolar bone and the tooth cementum .
previous studies showed that pdl cells play a role as producers of cytokines and chemokines in periodontal inflammation .
et-1 dose- and time - dependently induced the production of proinflammatory cytokines tnf- , il-1 , and il-6 by h - hpdl ; et-1 may be involved in the inflammatory process of periodontitis , at least in part , by stimulating proinflammatory cytokine production via the mapk pathway in hpdl cells .
pdlscs have a dynamic role in maintaining periodontal homeostasis and are responsible for remodelling and regeneration of periodontal tissues .
the primary aim of this study is to investigate how et-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells ( pdlscs ) from periodontitis patients .
therefore , the present study is designed to investigate the effects of et-1 on the differentiation and expression of proinflammatory cytokines such as tnf- , il-1 , and il-6 in pdlscs and put emphasis on exploring the possible roles of etr pathway , mapks pathway , wnt/-catenin pathway , and wnt / ca pathways in et-1-induced differentiation and cytokine expression .
after obtaining informed consent , pdlcs were isolated from the periodontal ligament tissues of periodontitis patients ( n = 3 referred to 3 independent cultures from different individuals ) .
periodontal ligament cells ( pdlcs ) were scraped from the middle third of healthy , noncarious premolar tooth roots ( n = 3 referred to 3 independent cultures from different individuals ) .
the cells were enzymatically digested for 45 min at 37c in 0.1% collagenase type i ( sigma - aldrich co. , st .
louis , mo , usa ) and maintained in alpha minimal essential medium ( mem ; gibco , life technologies co. , grand island , ny , usa ) , containing 10% fetal bovine serum ( fbs ; gibco ) , 200 mm l - glutamine ( invitrogen , life technologies co. ) , 100 u / ml penicillin , and 100 mg / ml streptomycin sulfate as the basic medium . the resultant cell suspension
was then seeded in 25 ml flasks ( falcon , bd biosciences , franklin lakes , nj , usa ) and incubated at 37c in humidified air with 5% co2 .
confluent cells were trypsinized and serially subcultured ; on the third passage , cells were stained with vimentin antibody for characterization .
human pdlscs were isolated using the cd146 microbead kit , with an ls column and a midimacs separator ( miltenyi biotec , bergisch gladbach , germany ) . in brief , pdlcs ( 1 10 cells ) were transferred into a 15 ml conical tube and centrifuged at 300 g for 10 min .
then , we checked the positive markers , cd73 , cd105 , and cd90 , and negative markers , cd34 , cd45 , cd14 , cd11b , and cd79a , by flow cytometry . before using pdlscs for experiments ,
the cultures were fixed in 4% formalin for 10 minutes , rinsed gently in pbs , and incubated in a humid chamber , first with blocking reagents for 1 hour and then with selected primary antibody vimentin ( santa cruz biotechnology ) , overnight at 4c for detection of markers for breast cancer .
the antibody was diluted ( 1 : 100 ) according to the manufacturer 's recommendations .
we used the standard protocol for the biotin - streptavidin staining kit ( santa cruz biotechnology ) .
the osteogenic potential of pdlscs was tested using alp staining . for alp staining , after induction , cells were fixed with 4% paraformaldehyde and stained with a solution of 0.25% naphthol as - bi phosphate and 0.75% fast red frv using an alp kit according to the manufacturer 's protocol .
tnf- , il-1 , and il-6 concentrations in cell - free supernatants were determined using enzyme - linked immunosorbent assay ( elisa ) kits ( r&d systems , minneapolis , mn ) according to the manufacturer 's protocols .
total proteins were extracted from cells by sonication in ripa buffer containing protease inhibitors ( roche ) .
after quantification of protein concentrations using the bca protein assay kit ( thermo scientific , rockford , il ) , the protein samples ( 20 g / lane ) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) and transferred onto polyvinylidene difluoride ( pvdf ) membranes ( millipore ) . after being blocked with 5% fat - free dry milk
, the membranes were incubated with anti - tnf alpha antibody ( ab66579 ) , anti - il1 beta antibody ( ab2105 ) , il-6 ( d3k2n ) rabbit mab , anti - gapdh antibody , runx2 ( d1l7f ) rabbit mab cst#12556 , anti - osteocalcin antibody ( ab93876 ) , anti - endothelin a receptor antibody ( ab76259 ) , anti - endothelin b receptor antibody ( ab39960 ) , phospho - p44/42 mapk ( erk1/2 ) ( thr202/tyr204 ) ( d13.14.4e ) xp rabbit mab cst#4370 , p44/42 mapk ( erk1/2 ) ( 137f5 ) rabbit mab ( cst 4695 ) , phospho - sapk / jnk ( thr183/tyr185 ) ( g9 ) mouse mab ( cst 9255 ) , sapk / jnk antibody cst#9252 , phospho - p38 mapk ( thr180/tyr182 ) ( d3f9 ) xp rabbit mab ( cst 4511 ) , p38 mapk antibody cst # 9212 , axin1 ( c95h11 ) rabbit mab cst#2074 , apc antibody cst # 2504 , gsk-3 ( 27c10 ) rabbit mab cst#9315 , -catenin ( l87a12 ) mouse mab cst # 2698 , tcf1 ( c63d9 ) rabbit mab cst2203 , lef1 ( c12a5 ) rabbit mab cst2230 , anti - dishevelled / dvl1 antibody ( ab106844 ) , anti - ip3kc antibody ( ab188007 ) , anti - camkii ( phospho - t286 ) antibody ( ab32678 ) , anti - nlk antibody ( ab97642 ) , and anti - p38 map kinase ( 341360 ) rabbit pab ( cst 506123 ) , respectively .
target bands were detected using a horseradish peroxidase - conjugated secondary antibody and developed using pierce enhanced cemiluminescencet ( ecl ) ( thermo scientific , fisher scientific , pittsburgh , pa , usa ) , followed by quantifying using the image quant tm las 4000 ( ge healthcare ) .
semiquantifications were performed using image j program ( national institutes of health , bethesda , md , usa ) .
total rna was isolated using total rna kit ( r6934 , omega bio - tech inc .
the relative levels of target gene mrna transcripts were determined by quantitative rt - pcr using the sybr green system and specific primers on the lightcycler system ( roche ) .
each pcr was performed in triplicate in a final volume of 20 l solution : 10 l of sybr green dye , 1 l of diluted cdna products , 0.2 m of each paired primer , and 8.6 l deionized water .
the pcr amplification was performed in triplicate at 95c for 10 minutes and was subjected to 40 cycles of 95c for 15 s , 60c for 30 s. the last cycle for dissociation of sybr green probe was 15 s at 95c , 30 s at 60c , and 15 s at 95c .
the relative levels of each gene 's mrna transcripts were calculated by normalizing to the gapdh and expressed as a relative ratio .
tests of equality of variance were carried out and post hoc tests were also performed .
the cultured cells were spindle - shaped ( figure 1(a ) ) and expressed a positive reaction to antibodies against vimentin ( figure 1(b ) ) .
streaming results ( figure 1(c ) ) combined with the morphologic and biologic characteristics and the site from which the sample was collected , these data confirmed that the cultured cells were pdlscs .
the staining showed that et-1 promoted differentiation of pdlscs into osteoblasts ( figure 2(a ) ) .
as shown in figure 2 , runx2 , ocn , and col1 mrna levels gradually increased with the exposure of et-1 , peaking at day 21 ( p < 0.05 ) . at each time point
( 7 d , 14 d , and 21 d ) , the expressions of runx2 ( figure 2(b ) ) , ocn ( figure 2(c ) ) , and col1 ( figure 2(d ) ) mrnas were induced at a dose - dependent manner ( 1 , 10 , and 100 nm ) .
the effect of et-1 on osteogenic differentiation of pdlscs was further verified by assessing the change in runx2 , ocn , and col1 expression of proteins associated with osteogenesis .
runx2 ( figures 2(e ) and 2(f ) ) , ocn ( figures 2(e ) and 2(g ) ) , and col1 ( figures 2(e ) and 2(h ) ) protein expressions levels were coincident with the mrna changes . to investigate whether et-1 could affect proinflammatory cytokine expression in pdlscs , tnf- ( figure 3(a ) ) , il-1 ( figure 3(b ) ) , and il-6 ( figure 3(c ) ) expression were measured by elisa . at each time point
( 12 h , 24 h , and 72 h ) , the secretion of tnf- , il-1 , and il-6 in pdlscs was induced at a dose - dependent manner ( 1 , 10 , and 100 nm ) .
also , the more secretion of proinflammatory cytokine was observed at the longer exposure time to et-1 .
the effects of et-1 on protein expression of tnf- ( figures 3(d ) and 3(e ) ) , il-1 ( figures 3(d ) and 3(f ) ) , and il-6 ( figures 3(d ) and 3(g ) ) in pdlscs were measured by western blotting . in agreement with the elisa results , et-1 induced tnf- , il-1 , and il-6 in a dose - dependent and time - dependent manner .
the stimulatory effects on protein expression reached a peak at 72 hours after stimulation . to examine signal transduction in et-1-induced differentiation and cytokine expression , endothelin a receptor ( etra ) and endothelin b receptor ( etrb ) mrna and protein expressions
were examined after 12 h , 1 d , 3 d , 7 d , 14 d , and 21 d 10 nm et-1 treatment .
the effect of et-1 on etra mrna expression level increased with increasing treatment time , peaking at 21 d ( figures 4(a ) and 4(d ) ) . a time - dependent increase in etra protein level
it was previously reported that p38 and erk1/2 mapks play important roles in the osteogenic differentiation pathway .
thus , we wondered whether p38 and erk1/2 mapks would be involved in et-1-induced osteogenic differentiation of pdlscs .
rna and proteins were extracted after pdlscs had been treated with 10 nm et-1 for 12 h , 24 h , and 72 h and 7 d , 14 d , and 21 d. and the levels of each protein ( p38 , phosphor - p38 , erk1/2 , phosphor - erk1/2 , jnk , and phosphor - jnk ) were measured .
rt - pcr showed no difference in erk , jnk , and p38 after et-1 treatment ( figures 5(a ) and 5(d ) ) .
western blot analysis indicated that exposure of the cells to et-1 effectively increased the levels of phosphor - erk1/2 , phosphor - jnk , and phosphor - p38 without altering the total amounts of these proteins .
this result indicated that activation of the mapk pathway correlated with osteogenic differentiation ( figures 5(b ) , 5(c ) , 5(e ) , and 5(f ) ) .
the canonical wnt/-catenin pathway is not only one of the most important signaling pathways governing bone homeostasis but also involved in the progression of osteoporosis .
thus , whether wnt/-catenin pathway would be involved in et-1-induced osteogenic differentiation of pdlscs was tested .
rna and proteins were extracted after pdlscs had been treated with 10 nm et-1 for 12 h , 24 h , and 72 h and 7 d , 14 d , and 21 d. and the levels of each mrna ( axin , apc , gsk3 , -catenin , and tcf / lef ) were measured .
upregulation of axin , apc , -catenin , and tcf / lef mrna levels was observed ( figure 6(a ) ) .
western blot analysis indicated that exposure of the cells to et-1 effectively increased the levels of axin , apc , -catenin , tcf , and lef , whereas it downregulated the phosphor - gsk3 ( figures 6(b ) and 6(c ) ) .
cisd2-deficient murine induced pluripotent stem cells showed dysregulation of osteogenic differentiation ; recent studies have indicated that cisd2 may be involved in ca homeostasis . therefore , we further explored whether wnt / ca pathway was involved in the differentiation of pdlscs promoted by et-1 .
rna and proteins were extracted after pdlscs had been treated with 10 nm et-1 for 12 h , 24 h , and 72 h and 7 d , 14 d , and 21 d. and the levels of each mrna ( dv1 , ip3 kinase c , camk , nlk , and p38 mapk ) were measured .
none of the above genes showed any change after et-1 treatment ( figure 7(a ) ) , nor did the protein levels ( figures 7(b ) and 7(c ) ) .
these results indicated that wnt / ca pathway was not involved in the differentiation of pdlscs promoted by et-1 .
periodontitis caused an increase in the proliferation of human periodontal ligament stem cells and a decrease in the commitment to the osteoblast lineage .
periodontitis is a globally prevalent inflammatory disease which causes the destruction of the periodontal structures ( i.e. , alveolar bone , periodontal ligament , and root cementum ) and potentially leads to tooth loss .
further , regeneration potential of endogenous pdlscs was impaired in periodontal ligament with chronic periodontitis .
pdlscs play an important role , not only in the maintenance of the periodontium , but also in promoting periodontal regeneration .
interestingly , pdlscs are a heterogeneous cell population , including cells at different stages of differentiation and lineage commitment .
previous studies demonstrated that et-1 can induce the production of il-1b , il-6 , and tnf- , which exert a variety of proinflammatory effects , from human bronchial epithelial cell lines and macrophages .
however , the mechanism by which et-1 regulates cytokine expression and differentiation in pdlscs has not been reported .
our results showed that et-1 promoted differentiation of periodontitis patients ' pdlscs into osteoblasts , and et-1 increased secretion of tnf- , il-1 , and il-6 in dose- and time - dependent manner .
the balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts , and an imbalance in this bone metabolism leads to osteoporosis .
moreover , et-1 increased expression of runx2 , ocn , and col1 , which are osteogenic markers .
there are currently two distinct human et receptors known as etra and etrb . in drug - induced gingival overgrowth ,
et-1 mrna expression was significantly higher in patients with csa - induced gingival overgrowth ( p < 0.001 ) than in patients with periodontitis and the controls .
etra mrna was expressed more than the etrb in all examined samples . to examine signal transduction in et-1-induced differentiation and cytokine expression , endothelin a receptor ( etra ) and endothelin b receptor ( etrb ) mrna and protein expressions
were examined after 12 h , 1 d , 3 d , 7 d , 14 d , and 21 d 10 nm et-1 treatment .
mitogen - activated protein kinases ( mapks ) are serine / threonine - specific protein kinases and play an important role in the intracellular signaling that occurs in response to extracellular stimuli .
mapks are divided into three subclasses : extracellular signal - regulated kinase ( erk1/2 ) , c - jun n - terminal kinase ( jnk ) , and p38 kinase .
erk1/2 is particularly activated by mitogens and growth factors acting through receptor protein tyrosine kinases or gq protein - coupled receptor agonists such as et-1 .
jnk and p38 are potently activated by cellular stresses , including oxidative stress and proinflammatory cytokines such as il-1 and tnf-. the mapks control the activity of many transcription factors .
jnk and p38 are associated with the immune response and p38 probably propagates an inflammatory response by , at least in part , increasing production of cytokines , including il-1 and tnf- .
the activation of wnt/-catenin pathway , which is a common signaling pathway regulating the cell differentiation , prevents skeletal aging and inflammation .
the canonical wnt/-catenin signaling pathway governs the lineage commitment of bipotential spc into osteoblasts or chondrocytes .
pathway analyses identify canonical wnt/-catenin pathway as another important mediator in et-1-induced osteogenesis . in conclusion
, we found that et-1 promoted differentiation of pdlscs into osteoblasts , and et-1 increased secretion of tnf- , il-1 , and il-6 in dose- and time - dependent manner .
et-1 promotes differentiation of periodontal ligament stem cells into osteoblasts through etr , mapk , and wnt/-catenin signaling pathways under inflammatory microenvironment . | periodontitis is a kind of chronic inflammatory disease that affects the tooth - supporting tissues .
et-1 is related to periodontitis and involved in the regulation of cytokines , but the mechanisms remain unclear . the aim of this study is to investigate how et-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells ( pdlscs ) .
pdlscs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with et-1 ( 1 , 10 , or 100 nm ) for 12 h , 24 h , or 72 h. the osteogenic potential of pdlscs was tested using alp staining .
tnf- , il-1 , and il-6 levels were evaluated by elisa and western blot .
runx2 , ocn , and col1 mrna and western levels were detected by rt - pcr and western blot , respectively . to examine the signaling pathways and molecular mechanisms involved in et-1-mediated cytokine expression and osteogenic differentiation , etr pathway , mapks pathway , wnt/-catenin pathway , and wnt / ca2 + pathway
were detected by rt - pcr and western blot , respectively .
et-1 promoted differentiation of pdlscs into osteoblasts by increasing secretion of tnf- , il-1 , and il-6 in a dose- and time - dependent manner .
et-1 also increased expression of runx2 , ocn , and col1 .
et-1 promotes differentiation of pdlscs into osteoblasts through etr , mapk , and wnt/-catenin signaling pathways under inflammatory microenvironment . |
mass spectrometry ( ms ) is currently the most commonly used technology for the identification and quantification of proteins . like in any other
omics field , the amount of data generated by ms - based proteomics has increased exponentially in the last few years , which has prompted the development of several data repositories .
the proteomics identifications database ( pride ) ( http://www.ebi.ac.uk/pride ) was developed at the european bioinformatics institute ( ebi ) , as a repository for the results of ms - based proteomics experiments , allowing data from a vast range of approaches , instruments and analysis platforms to be stored and disseminated in a common structured and queryable format .
originally established as a production service in 2005 , pride has previously been described ( 13 ) along with guidelines on using the database and its associated tools ( 46 ) .
pride does not stand alone in this field , however , as several other proteomics databases have been established over the past few years .
gpmdb ( 7 ) , peptideatlas ( 8) and proteinpedia ( 9 ) are among the most important representatives of these ( 10 ) .
additionally , the tranche system ( http://tranche.proteomecommons.org ) provides a data transfer layer relying on peer - to - peer internet protocol technology . finally , the most recently launched proteomics repository is the ncbi peptidome ( 11 ) , a centralized , public proteomics data repository not dissimilar from pride in its objectives . for an up - to - date review covering
pride stores three different kinds of information : ms and ms / ms mass spectra as peak lists , the derived peptide and protein identifications ( ids ) and any associated metadata .
indeed , one of the advantages pride offers over other proteomics databases lies in the amount of structured metadata it contains , which is a key requirement to put the stored data in biological and/or technical context .
furthermore , together with the newly released ncbi peptidome , the established pride database constitutes an actual structured data repository , and does not assume any editorial control over submitted data
. another important feature of pride is that it allows data to remain private while anonymously sharing it with journal editors and reviewers through so - called reviewer log - in accounts. as a result , pride is now the recommended submission point for proteomics data for several journals such as nature biotechnology ( 13 ) , nature methods ( 14 ) and proteomics ( http://www3.interscience.wiley.com/cgi-bin/jabout/76510741/2120_instruc.pdf ) .
two highly influential informatics tools have been developed in support of the pride database over the years : the ontology lookup service ( ols ) ( 15 ) and the protein identifier cross - referencing system ( picr ) ( 16 ) .
picr is used to map all submitted protein identifications in pride to all known accession numbers for these proteins in the most important protein databases , as well as some genomic ones ( 16 ) .
picr mappings are performed on the entire pride database at regular intervals in order to keep all accession numbers up - to - date .
the mappings allow accurate experiment - to - experiment comparison , even if the experiments relied on different sequence databases for the identifications , and since the mappings include an historical archive of identifiers , they also readily translate now defunctional accession numbers through time .
in addition to these two established tools , a new application called database on demand ( dod , http://www.ebi.ac.uk/pride/dod ) has recently been added to the pride toolkit ( 17 ) .
this tool allows custom sequence databases to be built in order to optimize the results from search engines for gel - free proteomics experiments ( 18 ) .
dod allows users to process and combine the uniprotkb / swiss - prot , uniprotkb / trembl and ipi databases using in silico protein maturation filters , serial enzymatic digests to reflect both in vivo and in vitro cleavages , amino- or carboxy - terminal ragging of sequences and mass and composition - based output filters , amongst others . in this article , we will , however , focus on the improvements made to the pride system over the last 2 years , and we will also highlight recently submitted datasets of interest .
data content in pride has increased substantially since the last pride nar database issue was published ( 3 ) . by 1 september 2009 ,
pride contained 9908 experiments ( compared with only 3185 when the last nar manuscript was submitted on september 2007 ) , more than 2.5 million identified proteins ( in contrast to 330 000 on september 2007 ; a 7.5-fold increase ) , 11.5 million identified peptides ( versus 2.1 million on september 2007 ; a 5.5-fold increase ) and 50.3 million spectra ( 2.6 million on september 2007 ; a 19-fold increase ) .
the increase in the total number of peptide ids ( 5.5-fold ) is reflected in the growth of the number of unique peptide sequences in the database , which have seen a 6.1-fold increase .
this latter observation is particularly interesting , as the identification of unique sequences typically levels off when applying repeat analyses in proteomics ( 18,19 ) .
it seems that it is primarily the diversity of data flowing into pride ( in terms of sample , experimental technique , instrument and search engine ) that allows the number of unique sequences to grow in lockstep with the total number of submitted peptide ids ( 20,21 ) .
the data included in the graph are number of spectra , protein identifications , peptide identifications and unique peptides .
the data included in the graph are number of spectra , protein identifications , peptide identifications and unique peptides .
note that these data holdings are absolute figures , not distinguishing public and private data . by 1 september 2009 , 8570 experiments in pride
the rest of the data are private since , as mentioned previously , pride allows data to remain confidential during manuscript review .
the complete set of data in pride comprises 60 species , including several model organisms ( table 1 ) .
animal species provide the majority of these data , since a total of 17 animal species are represented , contributing 84.4 and 74.3% of all protein and peptide ids in pride , respectively .
perhaps unsurprisingly , the largest amount of protein and peptide ids comes from human samples ( 38.1 and 35.7% of all protein and peptide ids in pride , respectively ) .
drosophila melanogaster ( 16.2 and 12.6% ) and mouse ( 14.8 and 9.0% ) are the next most popular data sources .
table 1.data content in pride split by taxonomic divisionsprotein ids ( % ) peptide ids ( % ) group of organisms ( number of species ) animals ( 17)84.474.3 plants ( 8)7.611.5 fungi ( 8)3.92.2 bacteria ( 20)2.611.6 others ( 7)1.50.4species homo sapiens38.135.7 d. melanogaster16.212.6 mus musculus14.89.0 a. thaliana6.711.0 danio rerio6.34.0 rattus
norvegicus3.63.1 c. elegans3.38.9 aspergillus niger1.50.3 s. typhimurium1.37.3 chlamydomonas reinhardtii1.30only the top 10 species in terms of protein and peptide identifications are shown .
data content in pride split by taxonomic divisions only the top 10 species in terms of protein and peptide identifications are shown .
bacteria are the most often represented group of organisms with 20 different species , among which salmonella typhimurium provides the largest number of protein and peptide ids ( 1.3 and 7.3% of all protein and peptide ids in pride , respectively ) .
additionally , eight different plant and fungal species are represented , thus constituting the second and third most popular group of organisms in pride ( table 1 ) . apart from the overall association of data in experiments , pride also has the concept of projects , which provide a way to organize several related experiments together in a hierarchical structure .
some of the most relevant datasets that have recently been made publicly available in pride are organized under such a project structure .
first of all , datasets belonging to the second phase of the hupo plasma proteome project ( ppp2 ) ( 22 ) are now present in pride .
the most complete and most thoroughly annotated dataset so far within the ppp2 project comes from richard smith s lab at pnnl ( pride accession numbers 81728544 , both inclusive ) ( 23,24 ) .
this set of experiments also constituted the first test case for a full proteomexchange ( 25 ) submission , which is explained in more detail in the last section of this article .
as already shown , pride is increasingly receiving submissions from species other than human and mouse .
in this context , pride now stores two large datasets ( accession number 9776 ; and accession numbers 97779794 , both inclusive ) that have been used to improve the annotation of the caenorhabditis elegans genome ( 26,27 ) .
interestingly , experiment 9776 is also the largest single experiment in pride , comprising > 85 gb of uncompressed information .
currently , the largest set of experiments in pride that belongs to the same project ( accession numbers 38667955 , both inclusive ) constitutes a quantitative analysis of the secretory pathway in rat ( 28 ) .
another very interesting dataset ( accession numbers 33213354 , both inclusive ) ( 29 ) describes a high - density , organ - specific proteome catalog generated from different organs , developmental stages and undifferentiated cultured cells from arabidopsis thaliana .
perhaps most strikingly , pride now also stores proteomics data from several extinct animals , including one dataset from tyrannosaurus rex ( accession number 8633 ) ( 30 ) , which is one of the most widely discussed proteomics datasets published to date ( see http://pubs.acs.org/action/showstorycontent?doi=10.1021%2fon.2008.11.21.172568 ) .
the main improvement in the pride web interface is the ability to store and display fragment ion annotations on tandem mass spectra .
as mentioned before , and described in detail in the next section , it has recently become possible for pride users to submit files containing fragment ion annotation directly , and subsequently visualize these in the online pride spectrum viewer ( figure 2 ) .
this feature has important implications for journal requirements relating to publication - associated proteomics data .
figure 2.screenshot showing spectrum fragmentation annotation on tandem mass spectra as visualized in the pride spectrum viewer. y ions are highlighted in red , b ions in blue and neutral loss ions in green .
screenshot showing spectrum fragmentation annotation on tandem mass spectra as visualized in the pride spectrum viewer. y ions are highlighted in red , b ions in blue and neutral loss ions in green . at present
, each journal essentially develops custom guidelines for data submission , which differ in scope and stringency .
the journal molecular and cellular proteomics ( mcp ) , an early adopter in terms of guidelines , has developed the so - called paris guidelines for reporting proteomics data ( 31 ) that include the requirement to provide annotated fragmentation spectra in several defined cases .
the fact that pride can now handle the submission and visualization of this type of spectral annotation ensures that submitters can achieve mcp compliance with ease .
another important development in the web interface is the seamless integration of picr mappings into pride queries , the venn diagram comparison tool and the pride biomart interface . as a result
memory of all identifiers ever used for a given protein in the majority of proteomics databases , enabling users to query by whichever accession number or identifier is most convenient for them .
furthermore , the venn diagram - based experiment comparison tool provides the most comprehensive tool available to the community to date to compare two protein lists . finally , the pride
identification detail view page has been thoroughly revised to reflect the picr mappings , along with a new view on the protein sequence that allows identified peptides and even post - translational modifications to be highlighted .
it is important to highlight here that pride is entirely dependent on data submissions by researchers , as detailed proteomics data can not be curated from existing literature .
the development of a new submission tool called pride converter ( 32 ) ( http://pride-converter.googlecode.com ) has been key in the large growth of data content in pride during the last year ( figure 1 ) .
pride converter has made submission to pride a simple and efficient process , since a submitter can now convert a wide variety of the most common proteomics data formats ( figure 3 ) directly to pride xml in eight easy steps in a user - friendly , wizard - like graphical user interface .
the resulting pride xml file is then automatically schema - verified , and can subsequently be submitted directly to the pride database .
one of the most recent features added to pride converter ( available from version 2.0 onwards , released in july 2009 ) is the handling of spectrum fragmentation annotation from mascot .dat files , omssa .omx files and ms_lims ( 33 ) . as a result , it is now trivial for users of these formats to submit and visualize spectrum annotations in pride , which also results in immediate compliance with the requirements of journals such as mcp .
it is important to note here that compliance in the past essentially comprised submitting hundreds or even thousands of pdf renderings of spectra to the journal , causing several logistical problems , as well as constituting a downright data loss , since spectra in this form are of course no longer machine - readable .
it allows the user to choose the appropriate format from the list of supported software - specific formats that can be converted into pride xml .
it allows the user to choose the appropriate format from the list of supported software - specific formats that can be converted into pride xml .
another very useful improvement in the pride submission process is the possibility to upload ( typically quite large ) pride xml files to an ebi ftp server ( 6 ) .
finally , it is important to highlight that there are no longer any file size limitations for submissions to pride ; indeed , as mentioned above , the largest single submission to pride currently stands at a pride xml file of > 85 gb .
pride is of course also a very interesting tool for large - scale data mining .
currently , the easiest way to do this is by using the pride biomart interface ( 34 ) . in the current biomart interface ( http://www.biomart.org/biomart/martview/ ) , it is possible to retrieve data from pride alone , but also to integrate information from pride with other resources . by september 2009 , pride data could be combined with data resources such as ensembl ( 35 ) , uniprot ( 36 ) , reactome ( 37 ) , interpro ( 38 ) , the macromolecular structure database ( msd ) ( 39 ) , the ipi database ( 40 ) and the rat genome database ( rgd ) ( 41 ) , but the number of resources is continuously growing .
as such , a complex query combining ms data from pride with genomic , structural or pathway data has become straightforward and very fast . this integrative way to access bioinformatics data will undoubtedly become even more powerful and increasingly important in the life sciences as information obtained through multiple omics technologies
is pieced together for a more complete picture of the underlying biology ( 42,43 ) . for even more sophisticated data mining purposes , users can also download the corresponding xml files for each experiment from the ebi ftp server ( ftp://ftp.ebi.ac.uk/pub/databases/pride ) , where these files are now available as gzipped files rather than the original zipped files .
the reason for this transition is that many pride xml files had grown larger than the maximal allowed file size for generic zip files ( which is 4 gb ) .
a final important improvement for the ftp files is that they now also contain all the protein mapping information from picr .
pride is a founding partner of the proteomexchange consortium , together with other important proteomics databases ( 25 ) .
the core members of this consortium ( pride , ncbi peptidome , tranche , peptideatlas and gpmdb ) are currently working on the implementation of a system that will allow proteomics data sharing between all the members , with pride and ncbi peptidome as the initial proteomexchange submission points .
draft guidelines for proteomexchange submissions are available ( http://www.proteomexchange.org ) , and a large - scale proteomexchange pilot submission has already been performed ( 6 ) . it must be noted here that it is not trivial to fulfil all the requirements for a proteomexchange submission .
this is the reason why , in addition to the proteomexchange initiative , pride and ncbi peptidome have formally agreed that they will replicate and share all their public data , again ensuring that data become optimally visible to the scientific community .
finally , at a different level of integration , pride data are now used in cross - references from uniprot ( available from uniprot release 14.6 , on december 2008 ) .
this allows pride submitters to dramatically improve the exposure of their data , and provides a first point of entry in using pride data to annotate uniprot protein entries .
we have here described how the pride database has evolved from its original role as a repository of proteomics identifications arising from ms data , to a knowledgebase that provides tools for complex queries and data retrieval , dataset comparison and access to additional automated annotation of submitted datasets .
there has been a huge growth in data content , which can be traced for two key developments : the new pride converter submission tool has made data submission much easier and more straightforward , and the fact that various journals in the field are now strongly supporting and even mandating deposition of proteomics data in proteomics repositories in general , and pride in particular . since the last pride nar database issue ( 3 ) , there have been other significant developments in the pride system .
one of the most important improvements is that it is now possible to submit files containing fragment ion annotations on tandem mass spectra and visualize these annotations in the pride spectrum viewer. in the same context , we are currently developing a pipeline to derive this information in a generic , automatic way for all the experiments already present in pride . at present , this automatic annotation feature is already visible for three experiments in pride ( accession numbers 13 , both inclusive ) and this form of automatic annotation will be extended to the rest of pride in the near future .
such a pipeline will be especially useful as there are several output file formats from search engines or proteomics pipelines that do not explicitly contain such information , for instance sequest and trans - proteomic pipeline ( tpp ) output files .
the current lack of generally practiced data sharing in the proteomics field has recently been addressed by an editorial in nature biotechnology ( 44 ) . in this context , the value of pride as a data repository was recently clearly proven as the public availability of a dataset from t. rex ( 30 ) , and enabled an ensuing healthy discussion in the proteomics community .
initiatives such as proteomexchange and the public data replication agreement between pride and ncbi peptidome are expected to help overcome the community s reticence about data disclosure .
however , it must also be taken into account that , mainly due to the existence of different data formats and the inherent complexity of the data to be exchanged , this data - sharing process is very resource - intensive and time - consuming for the data repositories . in order to overcome this situation ,
therefore , in the next version of the pride system ( version 3 ) , compliance with the hupo proteomics standards initiative s data standards mzml and mzidentml ( previously known as analysisxml ) will be ensured ( http://www.psidev.info ) .
another ongoing key development in pride that will benefit the proteomics community is the creation of a new database called pride - q ( for q - rated ) that will contain only the highest quality data from the pride repository .
the relationship between the current pride repository and the planned pride - q resource is very similar to the existing relationship between uniprotkb / swiss - prot and uniprotkb / trembl .
whereas uniprotkb / swiss - prot aims for assured quality and annotation , the uniprotkb / trembl aims primarily at fully capturing all available data .
this 5-year project , funded by the wellcome trust , started at the beginning of 2009 and will represent the first concerted effort to perform quality control analysis on proteomics data of such heterogeneity .
the prodac grant of the european union ( grant lshg - ct-2006 - 036814 ) ; wellcome trust ( grant wt085949ma ) . funding for open access charge : wellcome trust . | the proteomics identifications database ( pride , http://www.ebi.ac.uk/pride ) at the european bioinformatics institute has become one of the main repositories of mass spectrometry - derived proteomics data . for the last 2 years
, pride data holdings have grown substantially , comprising 60 different species , more than 2.5 million protein identifications , 11.5 million peptides and over 50 million spectra by september 2009 .
we here describe several new and improved features in pride , including the revised submission process , which now includes direct submission of fragment ion annotations . correspondingly , it is now possible to visualize spectrum fragmentation annotations on tandem mass spectra , a key feature for compliance with journal data submission requirements .
we also describe recent developments in the pride biomart interface , which now allows integrative queries that can join pride data to a growing number of biological resources such as reactome , ensembl , interpro and uniprot .
this ability to perform extremely powerful across - domain queries will certainly be a cornerstone of future bioinformatics analyses .
finally , we highlight the importance of data sharing in the proteomics field , and the corresponding integration of pride with other databases in the proteomexchange consortium . |
inflammation is recognized as a main pathogenic element in the development of pulmonary arterial hypertension ( ph ) [ 1 , 2 ] with several studies showing a significant association between systemic inflammatory markers and ph [ 3 , 4 ] .
inflammation is also a precursor for the development and progression of atherosclerosis and systemic hypertension ( shtn ) .
the association between c - reactive protein ( crp ) and shtn has been established in multiple cross - sectional studies [ 59 ] , in particular after the advent of high sensitivity crp ( hs - crp ) assays capable of detecting levels that were earlier considered to be normal .
higher levels of crp may contribute to the development of shtn through the reduction of endothelium - dependant relaxation by reducing nitric oxide production in endothelial cells [ 10 , 11 ] , resulting in vasoconstriction and increased production of endothelin 1 .
another marker of systemic inflammation is albumin which is an acute phase reactant that is influenced by multiple factors mainly including malnutrition and inflammation which may reduce albumin levels irrespective of the patient 's nutritional status [ 14 , 15 ] .
other inflammatory markers include erythrocyte sedimentation rate ( esr ) , ferritin , angiotensin - converting enzyme ( ace ) , and 25
oh vitamin d. sarcoidosis is a systemic inflammatory disease that is associated with higher incidence of ph not only in cases with evidence of advanced parenchymal fibrosis , but also in cases without significant interstitial lung disease .
in fact , anti - inflammatory therapies can have an impact on the progression of sarcoidosis - associated ph .
the relationship between chronic granulomatous inflammatory disease and systemic hypertension had not been previously explored .
the aim of this study was to investigate the relation between the degree of systemic inflammation measured by various inflammatory markers and shtn in sarcoidosis patients .
this is a retrospective observational study of consecutive adult subjects > 18 years who were diagnosed with sarcoidosis at the university of illinois at chicago between january 2010 and january 2015 .
the diagnosis of sarcoidosis was made according to the european respiratory society ( ers ) , american thoracic society ( ats ) , and world association of sarcoidosis and other granulomatous disorders ( wasog ) criteria .
the institutional review board of the university of illinois at chicago approved the study and waived the need for patient consent ( approval number of 20130195001 ) .
cases were divided into two groups based on the presence or absence of an underlying diagnosis of shtn .
the diagnosis of shtn was made according to the seventh report of the joint national committee guidelines ( jnc 7 ) if the blood pressure was 140/90 mmhg in at least two separate visits or if the patient has been taking antihypertensive medication(s ) .
the inflammatory markers evaluated in this study were serum esr , crp , albumin , ferritin , ace , and 25 oh vitamin d. patients had been routinely tested for these inflammatory biomarkers to track the treatment response in several visits during a year .
we used the mean recorded values for esr , crp albumin , ferritin , and 25 oh vitamin d. data on patient demographic characteristics , medical comorbidities , clinical and laboratory variables , pulmonary function test ( pft ) , treatment , and outcome were collected .
esr was read after 30 minutes of bed rest and the reference range for it is 020
the blood levels of crp were measured by immunonephelometry and the reference range for crp was 0.52
serum 25 ( oh ) vitamin d was assayed using a competitive protein binding assay employing automated chemiluminescence and expressed in ng / ml .
continuous variables are expressed as mean standard deviation or median and interquartile range ( iqr ) and compared using student t - test .
categorical variables are described as counts and percentages and compared using the chi - square test . in order to study the effect of increase esr , crp , and albumin level on the prevalence of shtn
, we have divided the cohort into 4 groups according to the 4 quartiles of the esr , crp , and albumin , and the cochran - mantel - haenszel common odds ratio test was used to evaluate the odds ratio ( or ) and p value for the prevalence of hypertension in the 2nd , 3rd , and 4th esr and crp quartiles using the first quartile as the comparator , or the 1st , 2nd , and 3rd quartile using the fourth quartile as a comparator in the case of albumin . chi - square test was utilized to detect whether the trend in shtn prevalence across the four esr and crp quartiles was significant or not .
a roc ( receiver operating characteristics ) curve analysis was implemented to detect the ideal cutoff value of esr , crp , and albumin that yields the highest sensitivity and specificity for predicting shtn and to calculate the area under the curve ( auc ) . in order to examine independent predictors for shtn in sarcoidosis patients and avoid confounding factors
the variables used in the model were either those that achieved statistical significance at p < 0.01 upon univariate analysis or variables considered to be clinically relevant . a p value less than 0.05 is considered statistically significant .
data were analyzed using ibm spss 21.0 statistical software ( ibm spss version 21.0 , armonk , ny ) .
a total of 108 cases diagnosed with sarcoidosis were included in the study , among which 55 patients ( 50.9% ) had shtn and 53 ( 49.1% ) were normotensive .
the mean age of the study population was 53.4 9.4 years , 76.9% were females , 70% were african americans , and
the results of univariate comparison of baseline demographics , clinical and laboratory characteristics , pft , echocardiographic data , and treatment in sarcoidosis subjects with and without shtn are summarized in table 1 . compared with normotensive patients , those with shtn
were older ( 56 versus 50.6 years , p = 0.002 ) , had a higher body mass index ( 33.5 versus 30.2 kg / m , p = 0.037 ) , had a higher proportion of african american race ( 89.1% versus 50.9% , p = 0.0001 ) , and had a longer mean duration of sarcoidosis ( 14.3 versus 10 days , p = 0.014 ) .
pulmonary - wise , subjects with shtn have a higher proportion of ph ( 37% versus 15.1% , p = 0.012 ) , were more likely to complain of dyspnea ( 62.7% versus 43.1% , p = 0.049 ) , and had significantly lower lung function values ( fvc% , fev1% , tlc% , and dlco ) .
most of the study cohort ( 83% ) was receiving oral steroids , 43.9% were on disease modifying antirheumatic drugs , 29.6% were on methotrexate , and 4.7% were on azathioprine . at 5 years ,
84 cases were still available for follow - up information while the rest were lost to follow - up . subjects with shtn had significantly higher mean and median esr levels ( table 2 ) .
furthermore , the prevalence of shtn significantly increased from the 1st to the 4th esr quartile , with crude prevalence rates of shtn being 10% , 40.9% , 63.2% , and 75% , respectively ( p value for the trend = 0.0001 ) .
a total of 23 subjects ( 67.6% ) in the shtn group had esr levels in the 3rd or 4th higher quartiles , compared with only 11 ( 32.4% ) of the subjects without shtn ( p = 0.002 ) . using the cochran - mantel - haenszel method , compared to the lowest esr quartile ,
the or to have shtn were 6.2 ( p = 0.034 ) , 15.4 ( p = 0.002 ) , and 33.8 ( p = 0.0001 ) for the 2nd , 3rd , and 4th esr quartiles , respectively .
roc curve analysis performed to detect the best cutoff value for esr in predicting shtn in sarcoidosis patients revealed an auc value of 0.795 ( 95% ci 0.6920.897 , p = 0.0001 ) ( figure 1 ) . a cutoff value for esr of 30
mm / hr yielded a 62% sensitivity and 80% specificity for predicting the presence of shtn in this sarcoidosis patient cohort .
subjects with shtn had a trend towards higher median and interquartile range of crp levels [ 1.1 ( 0.65 , 3.65 ) versus 0.9 ( 0.5 , 1.8 ) , resp .
, p = 0.067 ] compared with normotensive subjects as shown in table 2 . on dichotomizing crp values , sarcoidosis patients with shtn had a significantly higher frequency of crp > 2 mg / l ( p = 0.003 ) , crp > 2.5 mg / l ( p = 0.004 ) , crp > 3 mg / l ( p = 0.001 ) , and crp > 3.5 mg / l ( p = 0.01 ) .
when we dichotomized the cohort according to crp levels , the prevalence of shtn was 88.9% when crp > 3 mg / l versus 45% when crp < 1 mg / dl ( univariate or for the prevalence of shtn when crp > 3 mg / dl versus crp < 1 mg / dl 9.778 , 95% ci 1.98148.261 , p = 0.005 ) .
the crude prevalence of shtn increased from 30% in the first crp quartile to 76.2% in the 4th quartile ( or 6.933 , p = 0.006 ) .
p value for the trend in shtn prevalence across the 4 crp quartiles was 0.003 .
roc curve analysis for crp revealed an auc value of 0.644 ( 95% ci 0.5180.769 , p = 0.03 ) ( figure 1 ) and a cutoff value of 3 mg / l yielded a 37% sensitivity and 95% specificity for predicting the presence of shtn in this cohort of sarcoidosis subjects .
the median and interquartile range ( iqr ) of albumin for the entire cohort was 3.7 g / dl ( iqr 3.44 gm / dl ) .
sarcoidosis patients with shtn had a significantly lower mean albumin level compared with normotensives ( 3.44 versus 3.77 gm / dl , resp .
opposite to what was observed with esr and crp , the prevalence of hypertension significantly decreased along albumin quartiles ( 1st quartile < 3.4 , 2nd quartile 3.43.69 , 3rd quartile 3.74 , and 4th quartile > 4 gm / dl ) , with the crude prevalence rates of shtn being 70.8% , 52.6% , 48.3% , and 28.6% for the 1st , 2nd , 3rd , and 4th quartile , respectively ( p value for the trend = 0.025 ) .
using the cochran - mantel - haenszel method , the or to have shtn in the first albumin quartile compared with the 4th albumin quartile was 5.77 ( 95% ci 1.72519.287 , p = 0.004 ) .
roc curve analysis revealed an auc value of 0.670 ( 95% ci 0.5640.776 , p = 0.003 ) ( figure 2 ) . an albumin level < 3.3 gm
/ dl yielded a 35% sensitivity and 87% specificity for predicting systemic hypertension in this sarcoidosis cohort .
we have also evaluated the relationship between shtn and other nontraditional markers of inflammation including ferritin , ace , and 25-hydroxy vitamin d. on univariate analysis , there was no significant difference between those with shtn and normotensive subjects with regard to the mean ferritin level ( 245 versus 83 g / l , p = 0.262 ) , mean ace level ( 52.9 versus 71.4 u / l , p = 0.121 ) , and mean 25 oh vitamin d ( 15.7 versus 16.5 ng / ml , p = 0.657 ) .
multivariate logistic regression analysis with backward stepwise elimination did not change the positive dose - response relationship between the esr and crp levels and the prevalence of shtn . in the multivariate logistic regression model (
n = 70 , nagelkerke r = 0.332 , and p from hosmer and lemeshow = 0.775 ) , we found that having esr levels in the 3rd and 4th quartiles combined ( or 3.165 , 95% confidence interval 1.0449.593 , p = 0.042 ) and crp levels in the 4th quartile ( or 6.057 , 95% confidence interval 1.56723.415 , p = 0.009 ) remained independent predictors for a subject with sarcoidosis to have shtn after adjustment for age , sex , body mass index , dyslipidemia , sarcoidosis duration , the use of oral steroids , and hypoalbuminemia .
we have not found a relationship between other inflammatory markers such as ferritin , 25 oh vitamin d level , and neutrophil - to - lymphocyte ratio and shtn on univariate or multivariable analysis .
the variables used in the multivariate model are shown as a forest plot ( figure 3 ) .
we were able to show in this retrospective analysis that an independent relationship exists between the degree of systemic inflammation and shtn in sarcoidosis patients .
previous work showed that esr [ 19 , 20 ] and crp levels are higher in sarcoidosis patients compared to controls .
we observed that , among sarcoidosis subjects , esr and crp levels are almost twice as high in hypertensive subjects compared with normotensive patients ( 48.8 versus 23.2 mm / hr , p = 0.001 for esr , and 3.4 versus 1.7 , p = 0.067 for crp ) .
furthermore , the link between shtn and inflammation is supported by the stepwise increase in shtn prevalence across quartiles of both esr and crp .
these results imply that a more intense inflammatory response may have an important pathophysiologic contribution for the development of shtn in sarcoidosis .
patients with pulmonary sarcoidosis and systemic hypertension also had significantly higher frequency of pulmonary hypertension ( p = 0.012 ) where inflammation is a recognized factor in its pathogenesis .
the association between crp and shtn has been previously demonstrated in other studies not related to sarcoidosis . for example , in a cross - sectional study of 300 subjects 30 years old , bautista and colleagues found that the prevalence of shtn was 1.56 times ( 95% ci 1.142.13 , p = 0.005 ) higher in subjects in the fourth quartile of crp as compared to subjects in the first quartile . in a later cross - sectional survey of 8347 apparently healthy koreans ,
sung and colleagues found a significant positive association between the blood pressure and crp level ( p < 0.0001 ) and after adjustment for confounding variables , the prevalence of hypertension by crp was significantly higher in subjects in the second , third , and fourth quartiles of crp compared to subjects in the first quartile . furthermore crp was found to be a predictor of future development of hypertension . in an analysis of the women 's health study , 20,525 females with initially normal blood pressure were followed up for 7.8 years and crp was found to be significantly associated with an increased risk of developing shtn over time .
furthermore , magen and colleagues found a significant positive correlation between systolic blood pressure and crp levels , with significantly higher levels in the resistant hypertension group compared with the controlled hypertension group ( 6.9 5.8 versus 4.2 4.8 , p = 0.021 ) .
these studies utilized the hypothesis that inflammation is important in the pathogenesis of shtn . in our study
, we are trying to show whether systemic inflammation caused by other diseases such as sarcoidosis would predispose to the development of systemic hypertension just like it predisposes these patients to pulmonary hypertension irrespective of lung involvement . in the above mentioned studies , the association of shtn with elevated esr was either insignificant ,
however , we found that esr gives the highest auc value in predicting shtn compared with any of the inflammatory markers studies .
hypoalbuminemia has been studied in multiple respiratory and cardiac diseases and was found to be poor prognostic indicator and an independent predictor of mortality . a large prospective study on emergency department patients showed that the short - term mortality of patients with hypoalbuminemia was three times higher compared with patients with normal albumin after adjusting for several confounders .
albumin is a negative acute phase reactant that is influenced by mainly two factors : patient 's nutritional status and inflammation ; however , prior studies showed that inflammation reduces albumin levels irrespective of the patient 's nutritional state .
serum albumin level is reduced in acute and chronic inflammatory states due to increased degradation from the high catabolic rate in addition to its transudation to extravascular space from increased capillary permeability .
our experience is from a single center , the design is retrospective , and the cohort is relatively small due to rarity of the disease .
the main study limitation is that we have not taken into account whether the diagnosis of shtn was made before or after the diagnosis of sarcoidosis and therefore a proportion of these hypertensive patients may have had shtn prior to and independent of sarcoidosis .
however , this is very difficult to control for as both diseases ( hypertension and sarcoidosis ) are chronic diseases and to know the exact onset of either pathology is not possible .
for example , it is known that sarcoidosis patients with active disease have very high levels of esr and crp .
these inflammatory markers are also significantly elevated in sarcoidosis - associated arthritis , erythema nodosum , concomitant connective tissue disease , or simultaneous acute infections and these factors have not been accounted for .
we have only used shtn as a diagnosis without staging its severity and therefore we were unable to study the correlation between the level of these inflammatory markers and the severity of shtn .
the or for the prevalence of shtn across the quartiles is notably elevated because it is known that , in diseases with high prevalence , the or may overestimate the effect of exposure ( the prevalence of shtn in this cohort is 50.9% ) .
we conclude that the level of systemic inflammation in sarcoidosis patients reflected by a higher esr , crp may be associated with the presence of shtn .
this study provides insight of the role of systemic inflammation in the development of shtn as an additional complication of the disease , suggesting the importance of a closer follow - up of blood pressure in normotensive sarcoidosis patients with elevated inflammatory markers .
the study may also stimulate the conduction of further trials to assess the role of anti - inflammatory drugs in the control and/or regression of shtn in sarcoidosis subjects who had developed shtn after the diagnosis of sarcoidosis . | introduction . the association between the level of systemic
inflammation and systemic hypertension ( shtn ) among subjects with sarcoidosis has not been previously explored .
methods .
a retrospective study was conducted to investigate the relation between the level of systemic inflammation in sarcoidosis , measured by various serum inflammatory markers , and shtn
. results . among a total of 108 cases with sarcoidosis ( mean age :
53.4 years , 76.9% females ) , 55 ( 50.9% ) had shtn and 53 ( 49.1% ) were normotensive .
esr was highly associated with shtn .
the patients with shtn had higher mean esr levels compared with normotensives ( 48.8 35 versus 23.2 27 mm / hr , resp . ; p = 0.001 ) .
roc curve analysis for esr revealed an auc value of 0.795 ( 95% ci : 0.6920.897 ; p = 0.0001 ) . with regard to crp
, there was a trend towards higher mean values in shtn group ( 3.4 versus 1.7 mg / l ; p = 0.067 ) and significantly higher prevalence of shtn in the highest crp quartile compared to the lowest one ( 69.6% versus 30% ; or 4.95 ; p = 0.017 ) .
roc curve analysis for crp revealed an auc value of 0.644 ( 95% ci : 0.5180.769 ; p = 0.03 ) . on multivariate analysis , esr and the crp remained independent predictors for shtn among subjects with sarcoidosis .
conclusion .
systemic inflammation is associated with the presence of shtn in sarcoidosis . |
hepatitis b is one of the major health problems in the world and around two billion people are infected globally and most of them are asian . just like other developing countries , hepatitis b virus ( hbv )
it has been reported that 51 - 56% of iranian cirrhotic patients are hepatitis b surface antigen ( hbsag ) positive . in 1979
it was estimated that approximately one million hbsag positive carriers were living among 35 million iranians . in 1990s
rate of hbv infection was reported between 1.07 and 5% among blood donors or general population[3710 ] and in 2000s between 1 and 2% in different provinces.[1014 ] in fact , during the last two decades , obligatory infantile hbv vaccination with more than 95% of coverage has led to significant decline in rate of hbv infection in iran . in 2008 , in a systematic review of literature we gathered and pooled available data on seroepidemiology of hbv infection in general population of iran . the hbv infection prevalence in iran was estimated 2.14% ( 95% ci : 1.92 ; 2.35 ) . despite unreliable meta - analysis , its comprehensive literature review showed that , the data were available for only 7 out of 30 provinces of iran .
kurdistan is one of the provinces with missing data regarding seroprevalence of hbv infection . from public health view , in addition to seroepidemiology , the risk factors of hbv infection is of great important , since with proper intervention , hbv seroepidemiology can be contained .
the major known risk factors for transmission of hbv are from hbsag positive pregnancy , transfusion , hospitalization , tattoo , intravenous drug abuse and high risk sexual behaviors .
since the lack of information on hbv prevalence and distribution of its risk factors among the general population is an obstacle for formulating effective policies to reduce the burden viral hepatitis , this population based study was designed to determinate accurate estimate of hbv infection epidemiology and associated risk factors in kurdistan province .
kurdistan is a province with 54,592 km area and 2,880,312 populations that is located at western part of iran .
this province is of great importance since it has common border with iraq that have impaired health care system and lacks major health statistics including rate hbv infection .
the general population of kurdistan ( in the western of iran ) was studied [ table 1 ] .
temporal inhabitants of the household , non - iranian nationalities or those who did not consent to the study were excluded .
census information kurdistan and 9 studied districts demographic data of the study population the sample size was determined using [ deff*np(1-p)]/[(d / z1-/2*(n-1)+p*(1-p ) ] equation , when n is population size ( 1,440,518 ) , p is hypothesized % frequency of outcome factor in the population ( here 5% 2 ) , d is confidence limits as % of 100 ( absolute % , here 2% ) and deff is design effect for cluster surveys that was set to 1 .
one hundred clusters were selected from nine districts within kurdistan province with a cluster size of 20 .
postal code or family registry code was used to randomly select the first household for each cluster .
blood samples were obtained from each subject and a questionnaire was completed by a trained interviewer .
the questionnaire included demographic and anthropometric data and risk factors for hepatitis b. the blood samples were transferred to the regional laboratory .
after separation of serum from blood samples in local laboratory , serums were frozen in 20c and transferred to the central laboratory of iranian blood transfusion organization .
hbsag , hbcab , and anti - hdv antibody were evaluated using enzygnost hbsag , 5.0 kit ( dade behring , germany ) , hepanostica anti - hbc uni - form kit ( biomerieux , france ) , and ( eti - abdeltak-2 ; diasorin , dietzenbach , germany ) , respectively .
the study was approved by the institutional review board of the baqiyatallah university of medical sciences , baqiyatallah research center for gastroenterology and liver disease and iranian blood transfusion organization . written informed consent was obtained from all subjects before data collection .
continuous variables are presented as mean valuesstandard deviation ( sd ) , while qualitative and discrete variables are presented as absolute and relative frequencies in the form of percentage .
the 95% ci for rate of hbcab or hbsag positivity was calculated by mid p - exact method if npq 5 when n is sample size , p is the rate of positive cases and q is 1 ( rate of positive cases ) .
if npq had been 5 , then normal approximation method ( wald ) would have been used to make 95% ci .
the comparisons between continuous and qualitative variables were performed by student 's t - test .
multivariable logistic - regression analysis involving recorded risk factors and patients characteristics that had significant p value in univariate analysis of hbv serostatus was performed to identify independent predictors of positive hbv seromarkers .
a stepwise procedure was deployed with p = 0.05 as the threshold level for variables to be entered into and retained in the final model , and p = 0.1 as the threshold level for variables to be removed .
the general population of kurdistan ( in the western of iran ) was studied [ table 1 ] .
temporal inhabitants of the household , non - iranian nationalities or those who did not consent to the study were excluded .
the sample size was determined using [ deff*np(1-p)]/[(d / z1-/2*(n-1)+p*(1-p ) ] equation , when n is population size ( 1,440,518 ) , p is hypothesized % frequency of outcome factor in the population ( here 5% 2 ) , d is confidence limits as % of 100 ( absolute % , here 2% ) and deff is design effect for cluster surveys that was set to 1 .
one hundred clusters were selected from nine districts within kurdistan province with a cluster size of 20 .
postal code or family registry code was used to randomly select the first household for each cluster .
blood samples were obtained from each subject and a questionnaire was completed by a trained interviewer .
after separation of serum from blood samples in local laboratory , serums were frozen in 20c and transferred to the central laboratory of iranian blood transfusion organization .
hbsag , hbcab , and anti - hdv antibody were evaluated using enzygnost hbsag , 5.0 kit ( dade behring , germany ) , hepanostica anti - hbc uni - form kit ( biomerieux , france ) , and ( eti - abdeltak-2 ; diasorin , dietzenbach , germany ) , respectively .
the study was approved by the institutional review board of the baqiyatallah university of medical sciences , baqiyatallah research center for gastroenterology and liver disease and iranian blood transfusion organization .
continuous variables are presented as mean valuesstandard deviation ( sd ) , while qualitative and discrete variables are presented as absolute and relative frequencies in the form of percentage .
the 95% ci for rate of hbcab or hbsag positivity was calculated by mid p - exact method if npq 5 when n is sample size , p is the rate of positive cases and q is 1 ( rate of positive cases ) .
if npq had been 5 , then normal approximation method ( wald ) would have been used to make 95% ci .
the comparisons between continuous and qualitative variables were performed by student 's t - test .
multivariable logistic - regression analysis involving recorded risk factors and patients characteristics that had significant p value in univariate analysis of hbv serostatus was performed to identify independent predictors of positive hbv seromarkers .
a stepwise procedure was deployed with p = 0.05 as the threshold level for variables to be entered into and retained in the final model , and p = 0.1 as the threshold level for variables to be removed .
totally 81 , 5.02% ( 95% ci 4.03 ; 6.17 ) , and 13 , 0.80% ( 95% ci 0.44 ; 1.34 ) subjects from 1613 participants were hbcab and hbsag positive , respectively .
the rate of positive hbv seromarkers in various districts of kermanshah province is given in figure 1 .
geographical distribution of hbcab and hbsag seropositivity in various districts of kurdistan province hbcab seroprevalence ranged between 2 and 7% .
no case of hbsag positive was found in divandare , bijar , kamyaran and marivan . in sarv
in univariate analysis , there was no significant difference in the rate of hbsag ( p=0.1 ) or hbcab ( p=0.8 ) among various districts .
it can be seen that hbcab seroprevalence and the difference between males and females increase with age group .
seroprevalence of hbcab in male and female subjects in different age groups figure 2 shows univariate analyses of studied risk factors .
marital status , age and literacy were significant risk factors of hbcab or hbsag seropositivity .
risk factors associated with hbcab or hbsag in univariate analysis figure 3 shows multivariate analysis of risk factors with significant p values in univariate analyses .
age group 46 - 65 and marital status were independent predictors of hbcab or hbsag seropositivity .
hepatitis b virus ( hbv ) infection is a major cause of liver disease in the world . who has estimated that in more than 350 million people acute infection have turned to chronic infection .
it is also estimated that more than 500,000 deaths occur annually mainly of cirrhosis and hepatocellular carcinoma caused by chronic hbv infection .
monitoring of several scattered population based studies that have been carried out in different part of iran shows that hbv prevalence has decreased dramatically in iranian population during the last decade .
it is now considerably less than many developing countries and only slightly higher than that seen in industrialized countries such as the united states .
improvement of the people 's knowledge about hbv risk factors , national vaccination program from 1993 for all neonates , vaccination of high risk groups such as healthcare workers and the introduction of disposable syringes for use in vaccinations , hospitals and clinics might justify this decrease .
unfortunately there were no available data from kurdistan province so we could identify the trend of hbv epidemiology in the general population after introduction of universal hbv vaccination program .
our current survey shows that approximately less than 1% and around 5% of the general population in kurdistan province are chronic hbsag carrier and had previous exposure to hbv infection . we can estimate that 23,042 chronic hbsag carriers are living in this province and 144,016 have resolved hbv infection and some have occult hbv infection and are at greater risk of cirrhosis and hepatocellular carcinoma . in multivariate analysis
age is a common risk factor that almost is reported in all of seroepidemiologic studies of hbv infection .
the reason is that the risk and cumulative frequency of high risk behaviors increase with age and consequently increase the rate of hbv exposure .
10 and 1% of married subjects had positive hbcab and hbsag , respectively . therefore checking couples before marriage and vaccination are advisable since they can considerably decrease the prevalence of hbv infection in this province
our population based study suggests that intrafamilial hbv transmission plays a major role in hbv transmission in kurdistan province .
furthermore , approximately 5% of general population in this province has prior exposure to hbv and less than 1% is hbsag carriers . | introduction : hepatitis b virus ( hbv ) infection is a serious global public health problem affecting billions of people globally . the lack of information of its seroprevalence among the general population is an obstacle for formulating effective policies to reduce the burden viral hepatitis .
therefore , this population based serological survey was conducted in kurdistan province , where no epidemiological data was available to determine the prevalence and risk factors of hbv infection.methods:1613 healthy subjects were selected from all districts of kurdistan province ( in the western of iran ) using random cluster sampling .
the subjects age ranged from 6 to 65 years old .
serum samples were tested for hbcab , hbsag and anti - hdv antibody .
screening tests were carried out by the third generation of elisa .
various risk factors were recorded and multivariate analysis was performed.results:the prevalence of hbsag and hbcab in kurdistan was before 0.80% ( 95% ci 0.44 ; 1.34 ) and 5.02% ( 95% ci 4.03 ; 6.17 ) , respectively .
none of hbsag carriers had positive anti - hdv antibody .
predictors of hbsag or hbcab in multivariate analysis were : older age and marriage .
we did not find any significant differences between males and females.conclusion:our population based study suggests that intrafamilial hbv transmission plays a major role in hbv transmission in kurdistan province .
furthermore , approximately 5% of general population in this province has prior exposure to hbv and less than 1% is hbsag carriers .
however , we could not find any case of hdv infection among them . |
asthma affects over 300 million individuals worldwide , a figure that is estimated to grow by 100 million by 2025 . a chronic inflammatory disease of the airways , asthma has multifactorial pathophysiological causes and considerable heterogeneity in the classification of the disease by phenotype , aetiology , severity and interventional control .
current guidelines recommend stepwise management to gain and maintain control , in which the clinical definition of full control is daytime symptoms or use of reliever medication less than twice a week , no limitations of activity , no nocturnal symptoms and normal lung function .
furthermore , the american thoracic society and the european respiratory society state that any definition or measure of control must take into account the management of a patient s future risk .
thus , in clinical management of asthma , consideration must be given to reducing the frequency of exacerbations , preserving lung function , preventing reduced lung growth in children and minimising the adverse effects of any treatment .
for those receiving low - dose inhaled corticosteroids ( ics ) , current step - up treatment involves the addition of a long - acting 2-agonist ( laba ) or leukotriene receptor antagonist as controller therapy . in patients unable to attain or maintain control with ics and laba those in global initiative for asthma treatment steps 35
( figure 1)upward titration of ics dose , leukotriene modifiers , sustained - release theophylline , oral glucocorticosteroids and anti - immunoglobulin e ( omalizumab ) are all further or alternative treatment options . despite these guidelines and the wide range of therapies available , poor control of current asthma symptoms , and of future asthma exacerbations ,
continues to affect > 50% of patients , with exacerbations placing significant strain on their quality of life and on health - care systems .
risk factors associated with future exacerbations include previous exacerbations , poor control , inhaler technique and adherence , co - morbid allergic rhinitis , gastro - oesophageal reflux disease , psychological dysfunction , smoking and obesity .
the same factors , in addition to incorrect diagnosis , poor choice of inhaler , variation in individual treatment responses or genetic components , have been attributed to the underlying poor control .
there are a number of actions available in the primary care setting to reduce the impact of these factors ( figure 1 ) . in the light of such concerns around risk and poor control ,
a number of new therapies are under investigation , including long - acting anticholinergic bronchodilators ( the focus of this review ) , anti - prostaglandin d2 crth2 antagonists , phosphodiesterase-4 inhibitors , anti - leukotriene 5-lipoxygenase - activating protein antagonists and the monoclonal antibodies mepolizumab and lebrikizumab ( which are raised against interleukin-5 and interleukin-13 , respectively ) .
short - acting anticholinergic agents , particularly ipratropium bromide ( ipratropium ) and oxitropium bromide ( oxitropium ) , have been used in asthma for many years , although they have not become widespread because they are generally considered to be less effective than short - acting 2-agonists ( sabas ) for acute bronchodilation .
this , coupled with a perception that longer - term antagonism of cholinergic receptors induces little bronchodilation above that induced by labas , has meant that , in contrast to chronic obstructive pulmonary disease , long - acting anticholinergic bronchodilators have not been considered or thoroughly investigated as potential controller medication in asthma .
early studies demonstrated mild bronchodilation and protection , over 48 h , against methacholine - induced bronchoconstriction in male patients with asthma , and , in patients with severe persistent asthma , small improvements in lung function were observed with the laba salmeterol plus the long - acting anticholinergic bronchodilator tiotropium bromide ( tiotropium ) , with a halved dose of fluticasone propionate .
recently , phase i iii clinical investigation with long - acting anticholinergic bronchodilators in asthma has begun : two phase ii trials of umeclidinium bromide ( umeclidinium ) have completed ( nct01641692 ; nct01573624 ) , and phase ii and iii trials with tiotropium , as add - on therapy , have demonstrated improvements in lung function and a reduction in exacerbation risk in patients with poorly controlled asthma despite the use of ics or ics plus a laba . in this review ,
we consider the pathophysiological and clinical rationales for use of long - acting anticholinergic agents in the broader management of asthma , and the clinical evidence reported to date .
the symptoms of asthma , and of acute exacerbations , are attributed to airway narrowing that occurs as a consequence of chronic inflammation and associated hyper - responsiveness .
local influx of inflammatory cells and high levels of inflammatory mediators result in airway oedema , airway thickening , mucus hypersecretion and bronchial smooth muscle contraction ( table 1 ) .
although multiple pathophysiological mechanisms are thought to contribute to the characteristic narrowing of airways and the hyper - responsiveness found in asthma ( table 1 ) , bronchial smooth muscle contraction represents the primary cause of reversible airway obstruction in asthma .
tone ) is under autonomic nervous regulation ( figure 2 ) , although the mechanisms are not fully understood . during normal ventilation ,
adrenergic sympathetic nerves and parasympathetic cholinergic and non - cholinergic nerves are all active , but cholinergic activity is thought to be the predominant driver of bronchoconstriction ( figure 2 , box 2 ) .
acute treatment with the anticholinergic compounds atropine and ipratropium is known to reduce basal airway smooth muscle tone .
patients with asthma have increased basal airway smooth muscle tone , and there is evidence to suggest that this is a result of increased basal activity of pulmonary parasympathetic cholinergic nerves , hereinafter described as
.
demonstrated that bronchoconstriction induced by breath - holding is significantly inhibited by ipratropium in asthmatic patients but not in healthy volunteers .
it is thought that cholinergic tone , at least , is driven by afferent nervous activity arising in the airways , and it has been hypothesised that local airway inflammatory mediators may have a role in inducing afferent activity and an autonomic reflex response , thereby driving an increase in cholinergic tone ( figure 2 ) .
other proposed mechanisms for increased cholinergic tone in asthmatic patients include abnormal muscarinic receptor expression , increased release of acetylcholine from cholinergic nerve endings and reduced levels of neuromodulators that attenuate cholinergic neurotransmission . the degree to which cholinergic tone contributes to airway narrowing in asthma , either at basal state or during exacerbations , is unclear .
however , the fact that airway hyper - responsiveness can persist in asthmatic patients , possibly even in the absence of airway inflammation following long - term ics use , suggests that other pathophysiological factors , such as increased cholinergic and smooth muscle tone , have a role in asthma .
it has been proposed that acetylcholine has a prominent role in allergen - induced airway smooth muscle remodelling . in a guinea pig model of ongoing allergic asthma ,
treatment with tiotropium inhibited increases in airway smooth muscle mass and contractility induced by allergic challenge ; it has thus been hypothesised that anticholinergic drugs could help prevent airway smooth muscle remodelling in human asthma .
cholinergic activity is also believed to regulate non - smooth muscle and non - neuronal cells within the lungs , including inflammatory cells and those controlling mucus secretion . in a guinea pig model ,
tiotropium was shown to reduce allergen - induced mucus gland hypertrophy and goblet cell number , suggesting that anticholinergic bronchodilators might also reduce airflow obstruction by reducing mucus hypersecretion .
expression of cholinergic receptors on inflammatory cells raises the additional question of whether there are any non - neuronal anti - inflammatory actions of cholinergic antagonists , although a review of studies on chronic obstructive pulmonary disease failed to identify robust evidence of this .
anticholinergic bronchodilators are antagonistic to parasympathetic activity and exert their effects on acetylcholine receptors on airway smooth muscle and pulmonary parasympathetic nerves ( figure 2 ) .
acetylcholine receptors fall into two families nicotinic and muscarinic and it is the m1 , m2 and m3 subtypes of the latter that are thought to be primarily involved in the regulation of bronchoconstriction .
all subtypes of muscarinic receptors are widely expressed in the brain , the parasympathetic nervous system and the body s smooth muscle tissues .
m2 receptors are found in prejunctional membranes of neuromuscular junctions of airway smooth muscle and regulate negative feedback to reduce acetylcholine transmission . in a pulmonary context
, m3 receptors are predominantly expressed in smooth muscle cells , where they regulate contraction , and also within lung submucosal glands , where they regulate mucus secretion ( figure 2 ) .
thus , it is preferable for antimuscarinic bronchodilators to have a relatively high affinity for m1 and m3 receptors and low affinity for the m2 receptor . currently , there are five anticholinergic drugs available for bronchodilation in respiratory disease .
ipratropium and oxitropium are short - acting non - selective antagonists of m1 , m2 and m3 receptors .
in contrast , tiotropium , aclidinium bromide ( aclidinium ) and glycopyrronium bromide ( glycopyrronium ) are long - acting compounds , with comparative selectivity for the m1/m3 , m2/m3 and m3 receptors , respectively .
short - acting anticholinergics are generally considered less effective acute bronchodilators than sabas , and their short duration of action makes them broadly unsuitable as controller medication .
thus , evidence of increased cholinergic tone in patients with asthma indicates that the longer - acting bronchodilator compounds may be more suitable as controller medications in asthma .
there is some rationale to suggest that the addition of long - acting anticholinergic bronchodilators to labas might provide advantages in the treatment of asthma ( box 2 ) .
it is reasonable to hypothesise that by simultaneously antagonising parasympathetic smooth muscle contraction and stimulating adrenergic smooth muscle relaxation , it is possible to achieve greater bronchodilation compared with either strategy in isolation . to date , there has been little thorough clinical investigation of this hypothesis in asthma , but a study in a guinea pig model found that bronchodilation induced by the laba carmoterol was significantly augmented by the addition of tiotropium .
in vitro studies
have also found that the laba indacaterol can synergistically increase the inhibitory effects of glycopyrronium on methacholine - induced airway smooth muscle contraction . as discussed below , improvements in lung function have been observed in asthmatic patients receiving tiotropium as add - on therapy to laba plus ics .
it has been suggested that anticholinergic / laba combination therapy might offer advantages in mitigating daily variation , based on evidence that sympathetic activity may be elevated during the daytime , relative to the parasympathetic system , which may predominate at night .
for example , it was shown in a small study in patients with nocturnal asthma that ipratropium is more effective than salbutamol in the prevention of morning reductions in peak expiratory flow .
it is also possible that a combined approach to bronchodilation might reduce the impact of inter - patient variability in the relative responses to anticholinergic or adrenergic interventions .
finally , tachyphylaxis to the effects of -agonists is known to occur ( although the clinical relevance of this remains unclear ) , and it has been proposed that crosstalk between muscarinic receptor signalling and adrenergic receptor signalling in smooth muscle cells might interfere with tachyphylactic mechanisms .
this would provide a further rationale for the investigation of laba / long - acting anticholinergic bronchodilator combination therapy in asthma , as add - on to ics .
historically , short - acting anticholinergic bronchodilators have not been considered appropriate for the control of asthma , except in some cases for the acute treatment of asthma attacks in patients with chronic stable asthma , and in those who experience adverse events from sabas , such as tachycardia , arrhythmia and tremor .
although short - acting anticholinergics are considered less effective rapid bronchodilators than sabas such as salbutamol , there are data to suggest that , for acute exacerbations , ipratropium in combination with a saba as reliever medication improves lung function to a greater extent than a saba alone . in a double - blind ,
randomised trial , rodrigo and rodrigo investigated the effects of high - dose ipratropium plus the saba albuterol ( registered generic name for salbutamol in the usa ) in adults with acute asthma , in the emergency department .
patients receiving high - dose ipratropium plus albuterol had a greater improvement in peak expiratory flow and forced expiratory volume in 1 s compared with patients who received albuterol alone .
further , a meta - analysis has indicated that the addition of a short - acting anticholinergic to a saba is associated with a significant reduction in the risk of hospitalisation in children .
thus , in adults or children , the main justification for the use of short - acting anticholinergic drugs in acute asthma is reduction of the elevated airway smooth muscle and cholinergic tone during an acute crisis , although administration of multiple doses has been associated with a reduction in hospitalisations and risk of hospitalisation .
although tiotropium has been indicated for the treatment of chronic obstructive pulmonary disease for over a decade , no long - acting anticholinergic bronchodilators are currently approved in asthma .
a number of compounds exist , including aclidinium , glycopyrronium , glycopyrrolate and darotropium bromide , but , as mentioned , presently only tiotropium and umeclidinium have clinical trials in asthma listed on clinicaltrials.gov .
the latter has been under investigation in two dose - ranging phase ii trials in patients with asthma , as a monotherapy ( nct01641692 ) and in combination with fluticasone furoate ( nct01573624 ) , although to our knowledge no results from these trials have yet been published .
early studies with long - acting anticholinergics in asthma were small and underpowered , and failed to detect meaningful responses .
however , studies of tiotropium and of glycopyrrolate indicated that long - acting anticholinergics can provide sustained bronchodilation and bronchoprotection . to date , more thorough clinical evaluation has been performed with tiotropium only , in six phase ii or iii studies , involving over 3,500 patients ( table 2 ) . in an investigator - initiated three - way crossover trial
( 14 weeks per treatment ) in 210 patients with asthma inadequately controlled by low - dose ics ( twice - daily beclomethasone 80 g ) , tiotropium delivered via the spiriva handihaler device ( boehringer ingelheim pharmaceuticals , ridgefield , ct , usa ) was shown to be superior to a doubling of ics dose and equal to the addition of salmeterol , as assessed by improvements in lung function ( table 2 ) .
subsequent published investigations of tiotropium have all involved administration via the respimat softmist inhaler ( boehringer ingelheim pharma , ingelheim am rhein , germany ) . in an 8-week crossover trial , once - daily tiotropium at a dose of 5 or 10
g improved lung function , compared with placebo , in 107 patients with severe persistent poorly controlled asthma already receiving ics and laba ( table 2 ) . in a 16-week trial in patients with arginine / arginine homozygosity at amino acid 16 of the 2-adrenergic receptor ( b16-arg / arg ) and moderate poorly controlled asthma ( already receiving ics ) , once - daily tiotropium at a dose of 5 g was superior to placebo and non - inferior to twice - daily salmeterol at a dose of 50 g for maintenance of improvements in lung function ( table 2 ) . the rationale for performing the latter study
was based on suggestions that the adverse - event profile of 2-agonists is worse , and the efficacy lower , in patients with the b16-arg / arg polymorphism , although prospective investigation has revealed that there are no such concerns .
a subsequent phase ii dose - ranging study tested tiotropium at doses of 5 g , 2.5 g and 1.25 g as add - on to ics and found the 5 g dose to provide the greatest bronchodilator effect .
data from the first phase iii trial on a long - acting anticholinergic bronchodilator in asthma were published in 2012 . in two
replicate trials including a total of 912 patients with poorly controlled asthma despite the use of laba and high - dose ics ( 800 g budesonide or equivalent ) , tiotropium 5 g administered via the respimat softmist inhaler as add - on therapy significantly reduced the risk of severe exacerbations compared with placebo ( values provided in table 2 ) .
surprisingly , given the changes in lung function and exacerbation rate , improvements in symptomatic benefit ( seven - question asthma control questionnaire [ acq-7 ] and asthma quality of life questionnaire ) were small and inconsistent .
of adverse events reported in 2% of patients , only allergic rhinitis occurred at a statistically significantly higher rate in the tiotropium group compared with the placebo group .
dry mouth , a typical side effect associated with anticholinergic drugs , was reported in 2% of patients .
more recently , a phase iii replicate trial of once - daily tiotropium at a dose of 5 or 2.5 g , versus placebo , as add - on to medium - dose ics ( 400800 g budesonide or equivalent ) was conducted in 2,103 patients with poorly controlled asthma .
again , statistically significant improvements in lung function were observed with tiotropium , which were comparable in magnitude with those seen with salmeterol .
a statistically significant improvement over placebo in acq-7 responder rate was observed in all three active arms , although , as is common in analyses of acq-7 in asthma clinical trials , there was also a large placebo effect .
is it possible to determine to which patients , and in which clinical situations , long - acting anticholinergic bronchodilators might offer clinical benefits ?
phase iii investigation has found that tiotropium add - on therapy offers advantages to adults with severe asthma who are failing to gain control on ics and laba combinations .
this , and the fact that the benefit : risk ratio of ics falls at high ics doses , suggests that addition of long - acting anticholinergic bronchodilators to ics plus a laba is likely to be a useful option for patients with poorly controlled severe asthma , and an alternative to further increases in ics dose . whether long - acting anticholinergics will be appropriate as alternatives to labas is a harder question to answer .
nevertheless , tiotropium add - on to medium - dose ics has been shown to provide lung function and acq-7 improvements that were comparable with those of salmeterol , indicating that , in patients for whom labas may be unsuitable , long - acting anticholinergics could be a helpful alternative .
although the acq-7 effects reported in clinical trials thus far are relatively small , it will be interesting to see to what extent in practice patients gain clinically relevant benefits in control or future risk .
further , one might expect that the demonstrated reduction of exacerbation risk with tiotropium as add - on to ics plus laba might translate into long - term improvements in overall control .
we anticipate that lung function improvements of the magnitude observed in the trials we have described will translate into clinically relevant benefits to patients in a real - world setting . at the time of writing ,
few real - world studies have been performed , as long - acting anticholinergic bronchodilators are yet to be approved in asthma . however , in a retrospective study of the uk optimum patient care research database , off - label use of tiotropium in patients predominantly in global initiative for asthma step 3 or 4 was found to be associated with a reduction in the number of exacerbations and a reduced risk of severe exacerbation or lower respiratory tract infection .
it is yet to be determined in phase iii investigation whether long - acting anticholinergic bronchodilators offer similar benefits to adults with mild asthma or to children or adolescents , although several phase iii trials are underway with tiotropium in these populations ( nct01316380 ; nct01634139 ; nct01634152 ; nct01277523 ) .
there are some physiological ( box 2 ) and clinical rationales that allow us to suggest groups of patients for whom long - acting anticholinergic bronchodilators might be appropriate .
a few small studies with short - acting anticholinergic bronchodilators have indicated that responses to anticholinergics are more likely in older patients or in those with intrinsic ( non - allergic ) asthma .
it has also been suggested that patients intolerant of 2-adrenergic agents or with nocturnal asthma might respond better to anticholinergic bronchodilators .
further , there is evidence that patients with non - eosinophilic sputum profiles or neutrophilic inflammation do not gain the same benefit from ics as those with eosinophilic inflammation , and hence may be candidates for additional treatments such as long - acting anticholinergic bronchodilators , as may groups in which steroid resistance is known to occur , such as smokers or obese patients .
it is currently unclear why long - acting anticholinergic bronchodilators might reduce the rate of exacerbations .
however , one can hypothesise that a contributing factor to exacerbations might be an increase in afferent sensory nerve activity , resulting in an increase in parasympathetic tone and subsequent bronchoconstriction . if this were the case , treatment with long - acting anticholinergic therapies
it has long been apparent from clinical and preclinical investigations of the pathophysiology of asthma that cholinergic parasympathetic tone contributes to contraction of bronchial smooth muscle and narrowing of the airways .
the extent to which increased parasympathetic tone is a consequence of reflex to the inflammatory state or is a pathophysiological mechanism in itself is unclear .
regardless , the raised parasympathetic tone does provide a rationale for the use of long - acting anticholinergic bronchodilators in asthma , and recent phase iii trial results have demonstrated clinical benefits and lung function improvements with tiotropium as add - on therapy to ics alone or ics plus laba in adult patients with poorly controlled asthma . in light of the evidence , we believe that anticholinergic bronchodilators will be a useful add - on therapy for patients at high risk of future worsening or exacerbations , and in patients whose asthma remains uncontrolled on a broad range of treatments and/or for whom other alternative therapies are unsuitable .
whether tiotropium or other long - acting anticholinergic bronchodilators will offer clinical advantages in younger patients , or in those with less severe asthma than studied thus far , is under investigation .
as we gain clinical experience in asthma with long - acting anticholinergics , if approved , it will be interesting to see whether and to what extent certain subgroups and phenotypes benefit from their use as controller medications . | despite current guidelines and the range of available treatments , over a half of patients with asthma continue to suffer from poor symptomatic control and remain at risk of future worsening .
although a number of non - pharmacological measures are crucial for good clinical management of asthma , new therapeutic controller medications will have a role in the future management of the disease .
several long - acting anticholinergic bronchodilators are under investigation or are available for the treatment of respiratory diseases , including tiotropium bromide , aclidinium bromide , glycopyrronium bromide , glycopyrrolate and umeclidinium bromide , although none is yet licensed for the treatment of asthma .
a recent phase iii investigation demonstrated that the once - daily long - acting anticholinergic bronchodilator tiotropium bromide improves lung function and reduces the risk of exacerbation in patients with symptomatic asthma , despite the use of inhaled corticosteroids ( ics ) and long - acting 2-agonists ( labas ) .
this has prompted the question of what the rationale is for long - acting anticholinergic bronchodilators in asthma .
bronchial smooth muscle contraction is the primary cause of reversible airway narrowing in asthma , and the baseline level of contraction is predominantly set by the level of
cholinergic tone. patients with asthma have increased bronchial smooth muscle tone and mucus hypersecretion , possibly as a result of elevated cholinergic activity , which anticholinergic compounds are known to reduce .
further , anticholinergic compounds may also have anti - inflammatory properties .
thus , evidence suggests that long - acting anticholinergic bronchodilators might offer benefits for the maintenance of asthma control , such as in patients failing to gain control on ics and a laba , or those with frequent exacerbations . |
diabetes mellitus ( dm ) is a major global health problem . according to estimates of the world health organisation , there were 346 million people suffering from diabetes worldwide in 2011 .
the increased platelet activity is emphasized to play a role in the development of vascular complications of this metabolic disorder .
platelet volume , a marker of the platelet function and activation , is measured as mean platelet volume ( mpv ) by hematology analyzers .
diabetic patients have an increased risk of developing micro- and macrovascular disease , and platelets may be involved as a causative agent with respect to altered platelet morphology and function .
the aim of our study was to determine if platelets were activated in diabetes and in its associated vascular complications by measuring the mpv in the diabetics compared to the nondiabetics , to see if there was a difference in mpv in diabetics with and without vascular complications , and to determine the correlation of mpv with fasting blood glucose ( fbs ) , postprandial plasma glucose ( ppbs ) , glycosylated hemoglobin ( hba1c ) , body - mass index ( bmi ) , and duration of diabetes in the diabetic patients , respectively .
we also compared the mpv of diabetics having hba1c < 6.5% to that of diabetics having hba1c 6.5% .
this was a cross - sectional study carried out in 300 patients who were already diagnosed to have type 2 dm and 300 nondiabetic subjects without known coronary artery disease .
all the diabetic and nondiabetic subjects underwent a complete clinical evaluation with specific reference to any associated macro- or microvascular complications as well as any drugs taken .
we measured the mpv and platelet counts in the above target groups who had a complete blood count done using an automatic blood counter ( beckman coulter act5diff ) .
venous blood samples were collected in dipotassium edta and tested within 1 hour of collection to minimize variations due to sample aging .
samples for plasma glucose estimation and hba1c were collected in sodium fluoride and dipotassium edta , respectively .
the estimation of plasma glucose levels ( fasting plasma glucose and postprandial plasma glucose ) was carried out by the glucose oxidase method in the auto analyzer ( johnson and johnson vitros 250 ) and that of hba1c by the high - performance liquid chromatography method .
male patients with hemoglobin below 13 gm% and female patients below 12 gm% were excluded from the study because nutritional anemias can be a cause for reactive thrombocytosis and hence , increased mpv .
nondiabetic subjects with coronary artery disease and diabetics on antiplatelet drugs such as aspirin and clopidogrel were also excluded .
after baseline evaluation , diabetic patients were divided into two groups according to their hba1c levels : group a consisted of patients with hba1c levels < 6.5% and group b consisted of patients with hba1c levels 6.5% .
the latest hba1c cut - off for diabetic range according to american diabetic association 2010 criteria is 6.5% .
statistical evaluation was performed by statistical package for the social sciences ( spss ) version 14 ( chicago , il ) for windows statistics program using student 's independent sample two - tailed t - test and pearson correlation test ( r value as the coefficient ) .
among the 300 diabetic subjects in the study , 45 were excluded due to anemia or subjects on antiplatelet drugs .
similarly , among the 300 nondiabetic individuals , 49 were also excluded due to anemia or subjects who had history of coronary artery disease .
there were 166 male diabetics and 89 female diabetics in the study ( 255 in total ) .
there were 145 nondiabetic males and 106 nondiabetic females in the study ( 251 in total ) .
the mean age of the diabetic population was 5511.32 years , whereas that of nondiabetic population was 51.510.1 years .
out of the 255 diabetics , 159 ( 62.4 % ) had complications such as hypertension , peripheral neuropathy , autonomic neuropathy , diabetic foot , diabetic retinopathy , diabetic nephropathy , coronary artery disease , peripheral vascular disease , hypertriglyceridemia , and hypercholesterolemia and 96 ( 37.6 % ) did not have any of these complications .
kg / m whereas it was 24.664.2 kg / m in the nondiabetic group ( p=0.321 ) .
the mean fbs level in the diabetic population was 150.571.7 mg / dl while that of the nondiabetic group was 78.5613.15 mg / dl ( p < 0.001 ) .
the mean ppbs level in the diabetic population was 252.994.85 mg / dl while that of the nondiabetic group was 133.456.75 mg / dl ( p < 0.001 ) .
the mean hba1c level in the diabetic group was 9.132.53% as compared to 5.950.723% of the nondiabetic group ( p < 0.001 ) .
the mean platelet count in the diabetic group was 277.4681.13 10/l as compared to 269.7970 10/l of the nondiabetic group ( p=0.256 ) . in the diabetic subjects ,
mpv was significantly higher ( 8.290.735 fl ) as compared to the non - diabetic group ( 7.470.726 fl ; p < 0.001)[table 1 ] . among the diabetic subjects ,
a positive statistical pearson correlation was seen between mpv and hba1c levels ( r = 0.29 ; p < 0.001 ) , fbs levels ( r = 0.269 ; p < 0.001 ) and ppbs levels ( r = 0.194 ; p = 0.002 ) .
however , no statistical correlation was seen between mpv and the duration of dm , bmi and the vascular complications in the diabetic group [ table 2 ] .
comparison of various parameters between the diabetic and nondiabetic subjects correlation of mpv to the various parameters studied in the diabetic group , the mean mpv in subjects with complications ( 8.350.73 fl ) were higher than that of subjects without complications ( 8.20.74 fl ) but independent student t - test did not show any statistical significance ( p = 0.145 ) .
we also divided the diabetic group based on the hba1c levels into group a ( hba1c < 6.5% ) and group b ( hba1c 6.5% ) .
out of 255 dm patients , there were 34 patients in group a ( mean hba1c = 5.90.39% ) and 221 patients in group b ( mean hba1c = 9.632.35% ) .
the mean bmi in group a ( 23.62.94 kg / m ) was significantly lower than that of group b ( 25.244 kg / m ; p = 0.025 ) .
the mean fbs level in group a was 82.9719.99 mg / dl while that of group b was 160.971.15 mg / dl ( p < 0.001 ) .
the mean ppbs level in group a was 150.548.6 mg / dl while that of group b was 268.990.24 mg / dl ( p < 0.001 ) .
the mean platelet count in group a ( 289.467 10/l ) was higher than that of group b ( 275.683 10/l ) but was not statistically significant .
the mean mpv in group a ( 7.950.72 fl ) was significantly lower than that of group b ( 8.350.724 fl ; p = 0.003 ) [ table 3 ] .
dm is a complex metabolic syndrome characterized by chronic hyperglycemia resulting in complications affecting the peripheral nerves , kidneys , eyes , and micro- and macrovascular structures .
countries with the highest absolute number of diabetics are in india ( 19 million ) , china ( 16 million ) , and the united states ( 14 million ) .
the prevalence of diabetic microvascular complications is higher in people with poor glycemic control , longer duration of dm , associated hypertension , and obesity .
diabetes and its vascular complications can cause a financial havoc , become a burden to a country 's national economy and dent its growth .
mpv can be used as a simple economical test in the monitoring of dm and thereby help curb the morbidity and mortality . preventing vascular complications and monitoring of dm are the need of the hour as the prevalence of dm and its vascular burdens are increasing day by day .
type 2 dm is characterized mainly by impaired insulin secretion and increased tissue insulin resistance .
sustained hyperglycemia leads to a series of interrelated alterations that can cause evident endothelial dysfunction and vascular lesions in diabetic complications .
formation of advanced glycation end products , activation of protein kinase c and disturbances in polyol pathways are the possible mechanisms by which increased glucose induces vascular abnormalities .
primary plug formation due to platelets seals the vascular defects and provides the required phospholipid surface for the recruited and activated coagulation factors . in response to stimuli generated by the endothelium of blood vessels , platelets change shape , adhere to subendothelial surfaces , secrete the contents of intracellular organelles , and aggregate to form a thrombus .
these pro - aggregatory stimuli include thrombin , collagen , epinephrine , adp ( dense storage granules ) , and thromboxane a2 ( activated platelets ) .
thus , platelets may assume an important role in signaling of the development of advanced atherosclerosis in diabetes.[7911 ] mpv is an indicator of the average size and activity of platelets .
hence , they contain denser granules , secrete more serotonin and -thromboglobulin , and produce more thromboxane a2 than smaller platelets.[51113 ] all these can produce a pro - coagulant effect and cause thrombotic vascular complications .
this suggests a relationship between the platelet function especially mpv and diabetic vascular complications thus indicating changes in mpv reflect the state of thrombogenesis .
there might be small bleeds due to the rupture of atherothrombotic plaques leading to increased platelet recruitment , hyper reactivity , and bone marrow stimulation .
high mpv is emerging as a new risk factor for the vascular complications of dm of which atherothrombosis plays a major role .
platelet hyperactivity has been reported in diabetics and animals , both in vivo and in vitro .
if vascular damage was only due to increased number of large and reactive platelets , then the rate of damage would have been constant for the duration of disease and independent of diabetic control .
this clearly shows that platelet reactivity alone can not explain the progression of vascular complications in dm since there are other vascular risk factors that may be influenced by the degree of control of diabetes .
this was supported by the nonsignificant statistical correlation between mpv and duration of diabetes in the study .
a direct relation between platelet dysfunction and the development of diabetic complications has yet to be firmly established .
platelet hyper - reactivity and increased baseline activation in patients with diabetes is multifactorial.[111619 ] it is associated with biochemical factors such as hyperglycemia and hyperlipidemia , insulin resistance , an inflammatory and oxidant state and also with increased expression of glycoprotein receptors and growth factors.[111619 ] hyperglycemia can increase platelet reactivity by inducing nonenzymatic glycation of proteins on the surface of the platelet , by the osmotic effect of glucose and activation of protein kinase c.[1618 ] such glycation decreases membrane fluidity and increases the propensity of platelets to activate.[1618 ] platelet function is directly regulated by insulin via a functional insulin receptor ( ir ) found on human platelets.[1618 ] in vivo experiments have confirmed that insulin inhibits platelet interaction with collagen and attenuates the platelet aggregation effect of agonists in healthy nonobese individuals.[1618 ] in inflammation , superoxide increases intraplatelet release of calcium after their activation and thus enhancing platelet reactivity.[1618 ] furthermore , superoxide limits the biologic activity of nitric oxide ( no ) because the oxidative stress impairs endothelial function that reduces production of no and prostacyclin.[1618 ] decreasing the effect of no brings about increased platelet reactivity.[1618 ] platelets from patients with diabetes express more surface p - selectin and glycoprotein ( gp ) iib / iiia receptors and are more sensitive to agonist stimulation than platelets from patients without diabetes .
platelets in dm have dysregulated signaling pathways that lead to an increased activation and aggregation in response to a given stimulus ( platelet hyper - reactivity ) .
platelet activation contributes to the pathology by triggering thrombus formation and causing microcapillary embolization with the release of constrictive , oxidative , and mitogenic substances such as platelet - derived growth factor ( pdgf ) and vascular endothelial growth factor ( vegf ) that accelerate progression of local vascular lesions like the neovascularization of lens in diabetic retinopathies . at the same time
, mpv can also be elevated as an end result of an atherothrombotic event like myocardial infarction .
this could be due to the quicker consumption of smaller platelets in the vascular event and compensatory production of reticulated platelets . in our study ,
the mean platelet count in the diabetic group was higher than that of the nondiabetic group that was similar to the studies done by demirtunc et al . and zuberi et al .
other studies by hekimsoy et al . had observed the opposite finding with lower platelet counts in the diabetic group compared with nondiabetic healthy subjects .
hence , the platelet count could be dependent on several variables , that is , mean platelet survival , platelet production rate , and turnover rate in dm . in our study ,
, demirtunc et al . , zuberi et al . , atea et al . , jindal et al . , and papanas et al.[46121422 ] in our study , higher values of mpv were observed in diabetic subjects with microvascular complications such as retinopathy and microalbuminuria but were not statistically significant .
higher values were also seen in the studies done by ates et al . and papanas et al .
this suggested a role for the increased platelet activity in the pathogenesis of vascular complications . on the other hand , in the studies done by hekimsoy et al . and demirtunc et al .
mpv was not significantly different in subjects with diabetic neuropathy / retinopathy from that of diabetics without those complications .
their possible explanation was centered on the rapid consumption of activated platelets in diabetics with complications . in our study , mpv was significantly higher in diabetics with hba1c levels 6.5% than in diabetics with hba1c levels < 6.5% .
there was also a significant association between hba1c and mpv , which was again seen in the study done by demirtunc et al .
therefore , it may be concluded that glycemic control decreases the hyper activity of the platelet function and thus may prevent or delay possible diabetic vascular complications .
the reason for a high number of diabetics with hba1c levels 6.5% in the current study ( 221 ) might have been due to poor dietary practices and lack of knowledge regarding the diet and exercise regimens that ought to be followed in diabetics .
no mpv association was seen with duration of diabetes , bmi and presence of complications .
but our findings were in contrast to the study done by ates et al . where mpv was positively correlating with the degree of retinopathy in their cases
. the limitations of the study that could not be assessed were qualitative platelet disorders and other reactive causes for raised platelets like menstruation that constitute a minor role .
our study showed that in diabetes mellitus , platelets become more reactive and aggregable and their mean volume ( mpv ) is increased .
the increased platelet size may be one factor in the increased risk of atherosclerosis associated with diabetes mellitus and associated vascular complications .
hence , mpv would be a useful prognostic marker of cardio - vascular complications in diabetes .
however , the increased mpv as the cause or the end result of vascular complications needs to be further explored .
hence , we propose that mpv can be used as a simple and cost - effective tool to monitor the progression and control of dm and its cardio - vascular complications . | context : diabetes mellitus is a global pandemic . the increased platelet activity may play a role in the development of vascular complications of this metabolic disorder .
the mean platelet volume ( mpv ) is an indicator of the average size and activity of platelets .
larger platelets are younger and exhibit more activity.aims:to determine the mpv in diabetics compared to nondiabetics , to see if there is a difference in mpv between diabetics with and without vascular complications , and to determine the correlation of mpv with fasting blood glucose , glycosylated hemoglobin ( hba1c ) , body - mass index , and duration of diabetes in the diabetic patients.materials and methods : platelet counts and mpv were measured in 300 type 2 diabetic patients and 300 nondiabetic subjects using an automated blood cell counter .
the blood glucose levels and hba1c levels were also measured .
statistical evaluation was performed by spss using student 's t test and pearson correlation tests.results:the mean platelet counts and mpv were higher in diabetics compared to the nondiabetic subjects [ 277.46 81 x 109/l vs. 269.79 78 x 109/l ( p= 0.256 ) ] , 8.29 0.74 fl versus 7.47 0.73 fl ( p= 0.001 ) , respectively .
mpv showed a strong positive correlation with fasting blood glucose , postprandial glucose and hba1c levels ( p=0.001).conclusions : our results showed significantly higher mpv in diabetic patients than in the nondiabetic subjects .
this indicates that elevated mpv could be either the cause for or due to the effect of the vascular complications .
hence , platelets may play a role and mpv can be used as a simple parameter to assess the vascular events in diabetes . |
dna double - strand breaks ( dsbs ) can be accidentally introduced in cells by the action of ionizing radiation or certain reactive radicals .
these agents have the ability to initiate a series of chemical reactions that ultimately sever the dna backbone , resulting in chromosome breakage and fragmentation of genes ( hoeijmakers , 2001 ) . because such corruption of genetic material inevitably leads to problems with replication and cell division , it is of the utmost importance that cells have a mechanism to counteract dsbs .
in addition , dsbs are generated in developing b and t cells during normal v(d)j recombination , implying that a working dsb repair system is not only necessary for an effective defense against dna - modifying agents but also for a functional immune system in higher organisms ( for review see weterings and van gent , 2004 ) . as a result ,
two highly efficient dsb repair pathways have evolved in eukaryotic cells : homologous recombination ( hr ) and nonhomologous end joining ( nhej ) .
( 3 ) processing of dna ends . ( 4 ) ligation of dna ends .
the hr process mediates dsb repair by using a homologous dna sequence as a template to guide proper restoration of the break .
because homologous templates are found on sister chromosomes , hr is thought to be active during the s and g2 cell cycle phases .
contrasting , nhej is characterized by its ability to directly ligate the two ends of the broken dna molecule .
this process does not have the need for a homologous template and is therefore theoretically not restricted to a certain phase of the cell cycle .
the nhej pathway utilizes several enzymes that capture both dna ends , bring them together in a synaptic complex , and facilitate direct ligation of the dna break ( fig .
the process is initiated by the association of dna ends with the ku 70/80 heterodimer , a protein with a ring - shaped structure that displays an extraordinary affinity for open dna ends ( walker et al . , 2001 ) . the dna
ku 70/80 complex then functions as a scaffold to assemble the other key nhej proteins at the dna termini .
scaffold is a large , 460-kd serine / threonine kinase called dna - dependent protein kinase catalytic subunit ( dna - pkcs ) .
the protein complex that is formed after the association of both ku 70/80 and dna - pkcs at the dna ends is generally referred to as dna - dependent protein kinase ( dna - pk ) .
the large dna - pkcs molecule forms a distinct structure at the dna termini , which is likely to play an active role in the formation of a synaptic complex that holds the two ends of the broken dna molecule together ( fig . 1 ; for review see weterings and van gent , 2004 ) .
the positioning of the two dna ends in a synaptic complex sets the stage for religation of the broken dna molecule . before this can take place effectively ,
it is necessary that noncomplementary ends are processed . in the case of single - stranded overhangs ,
dna termini can be made ligatable by either filling of the missing nucleotides or by resection of the overhang .
polymerases and , terminal deoxynucleotidyltransferase , polynucleotide kinase , and several nucleases have been shown to play a role in this processing .
the best - characterized processing nuclease is the endonuclease artemis , whose activities include the removal of single - strand overhangs ( ma et al . , 2002 ) .
xrcc4 complex , possibly in conjunction with the recently discovered xlf ( xrcc4-like factor)/cernunnos protein ( ahnesorg et al . ,
2006 ) , although at this point , little is known about the mechanism by which xlf stimulates ligation .
after the introduction of a dsb , the dna - pkcs enzyme is quickly recruited to the dna ku scaffold .
the serine / threonine kinase activity of dna - pkcs is not activated until the dna - pkcs molecule is associated with both the ku 70/80 heterodimer and a dna terminus .
many targets for the dna - pkcs kinase have been identified in vitro , including xrcc4 , ku 70/80 , artemis , p53 , and even dna - pkcs itself ( autophosphorylation ) .
this plethora of possible targets suggests that dna - pkcs may be involved in several aspects of the nhej process , including activation of components of the synaptic complex and signal transduction to cell cycle regulators ( burma and chen , 2004 ) .
surprisingly , however , little evidence has been found for the biological relevance of these activities . to the contrary ,
much more convincing data are available that argue for a regulatory role for dna - pkcs at the synapsis of the two dna ends during nhej .
dna - pkcs complex is scarce and provides little detail as a result of technical difficulties in the production of dna - pkcs crystals for crystalographic structure studies . the best available information on the three - dimensional structure of the dna ku dna - pkcs heterotrimer was obtained by single- particle electron microscopy at a 25- resolution ( spagnolo et al . , 2006
these images clearly showed dimeric structures in which dna ends were brought into close proximity by the formation of a synaptic complex that consisted of two dna ends , two ku 70/80 molecules , and two dna - pkcs molecules ( fig .
, both atomic force microscopy studies and biochemical experiments have shown that dna - pkcs not only assembles at dna ends but also facilitates the tethering of multiple dna ends ( for review see weterings and van gent , 2004 ) .
collectively , these findings suggest that dna - pkcs is responsible for the formation of a synaptic bridge between the two termini of the broken dna molecule .
it has been well established that the presence of the large dna - pkcs molecule at dna ends effectively blocks access of either processing nucleases or ligases ( weterings et al . , 2003 ; block et al . , 2004 ) .
although this capping of dna ends may have an important function in protection of the dna termini against degradation or premature and incorrect ligation , it is clear that the dna - pkcs cap has to be removed or altered before religation of the dna ends and repair of the dsb can take place .
several authors have demonstrated that autophosphorylation of dna - pkcs results in release of the cap and accessibility of the dna termini for either processing enzymes or ligases ( ding et al . , 2003 ; weterings et al . , 2003 ;
recent research has demonstrated that dna - pkcs autophosphorylation can occur in trans during the synapsis of two dna - bound dna - pkcs molecules ( meek et al . , 2007 ) .
these findings clearly argue for a model in which dna - pkcs protects the termini of a broken dna molecule by capping it until the dna ends find each other and are properly aligned in a synaptic complex .
dna - pkcs trans - autophosphorylation then liberates the dna ends for proper processing and ligation by other nhej factors like artemis ( goodarzi et al . , 2006 ) and ligase iv
dna - pkcs functions as a gatekeeper of the nhej process , regulating access to the dna ends by autophosphorylation ( fig .
, it is not clear how we should envision the conformational changes in the dna dna - pkcs complex that take place during autophosphorylation .
it is also not unequivocally known which autophosphorylation sites need to be phosphorylated to facilitate the accessibility of dna termini .
thus far , 16 amino acid residues that can be phosphorylated by the action of the dna - pkcs kinase have been identified within the dna - pkcs molecule ( fig .
the importance of dna - pkcs autophosphorylation during nhej is demonstrated by numerous observations , showing that mutation of phosphorylation sites causes increased radiosensitivity and less efficient dsb repair ( chan et al . , 2002 ; ding et al . , 2003 ; chen et al . , 2005 ; douglas et al . , 2007 ) and that an active dna - pkcs kinase is required for nhej ( kurimasa et al . , 1999 ) .
dna - pkcs protects the dna ends until trans - autophosphorylation induces a conformational change that enables dna end processing and ligation .
in addition to autophosphorylation , atm - mediated phosphorylation of dna - pkcs may play a role in this process . inhibiting phosphorylation of the entire 2609 cluster ( fig .
3 ) leads to a severe increase in radiosensitivity and diminished processing of dna ends , whereas mutation of single residues within this cluster has a less severe ( but still present ) effect on radiosensitivity ( chan et al . ,
in addition , we found that mutation of either the dna - pkcs kinase domain or the 2609 cluster and the 2056 residue results in a rigid binding of dna - pkcs to dna ends in vivo , which most likely interferes with the nhej process ( uematsu et al . , 2007 ) .
collectively , these results demonstrate that autophosphorylation of the 2609 cluster influences the dna dna - pkcs interaction in a manner that facilitates end joining .
3 ) has been reported to inhibit dna end processing upon phosphorylation , suggesting that the 2609 and 2056 clusters work in an opposite direction ( cui et al . , 2005 ) .
the latter finding has at present not been confirmed , and it is not entirely transparent how this double regulation mechanism cooperates during dsb repair .
phosphorylation of the recently discovered 3950 residue , which is located in the c - terminal kinase domain , most likely plays a role in regulation of the dna - pkcs kinase activity .
mutation of this site with phosphomimic aspartic acid results in deficient v(d)j recombination and increased radiation sensitivity ( douglas et al . , 2007 ) .
this finding suggests that the 3950 residue may be involved in the regulation of dna - pkcs autophosphorylation by mediating kinase activity .
three other autophosphorylation sites have been identified in the c - terminal region of dna - pkcs ( 3821 , 4026 , and 4102 ) , although no in vivo functionality has been reported for these sites at present ( ma et al . , 2005 ) .
recent experiments have shown that in vivo phosphorylation of the dna - pkcs 2609 and 2647 residues can still occur at dsb sites in the absence of dna - pkcs kinase activity ( chen et al .
, 2007 ; uematsu et al . , 2007 ) . clearly , in these cases , the dna - pkcs molecule is not responsible for the phosphorylation events , and another kinase ( or kinases ) must be involved .
these studies raise doubts as to whether all phosphorylation sites within the dna - pkcs molecule are genuine autophosphorylation sites or whether they can be targets for other kinases as well .
the 2609 cluster ( chan et al . , 2002 ; douglas et al . ,
2002 ) , 2056 cluster ( chen et al . , 2005 ; cui et al . ,
2005 ) , 3205 residue ( douglas et al . , 2002 ) , 3950 residue ( douglas et al . , 2007 ) , and 3821 , 4026 , and 4102 residues ( ma et al . , 2005 )
one study reports that phosphorylation of the 2609 and 2647 residues ( but not the 2056 residue ) of dna - pkcs is dependent on the ataxia telangiectasia mutated ( atm ) protein ( chen et al . , 2007 )
atm is a damage - responsive protein kinase and a member of the phosphatidylinositol 3-kinase like kinase group ( pikk ) , to which dna - pkcs also belongs .
another study finds no absolute requirement for the presence of atm but does acknowledge that several dna - pkcs residues can be phosphorylated in the absence of a functional dna - pkcs kinase , leaving open the possibility that atm contributes to the phosphorylation of dna - pkcs residues in vivo ( meek et al . , 2007 ) .
a third study suggests that yet another member of the pikk group , ataxia telangiectasia related protein ( atr ) , is involved in the regulation of dna - pkcs phosphorylation at the 2609 and 2647 residues after the onset of uv damage ( yajima et al . , 2006 ) . at present , it is not clear whether the atr - mediated phosphorylation of dna - pkcs is relevant for dsb repair via the nhej pathway . at this point , it is not known whether atm ( or atr)- mediated phosphorylation of the dna - pkcs 2609 and 2647 residues is redundant with autophosphorylation , but it is tempting to speculate that both routes of dna - pkcs phosphorylation have their own biological significance .
atm is activated at an early stage after the introduction of a dsb , is present at dsb sites , and is involved in signal transduction and phosphorylation of a plethora of proteins that mediate cell cycle arrest during dsb repair ( lobrich and jeggo , 2005 ) .
it is not unlikely that the atm kinase , with its central role in the regulation of dsb repair and its physical presence at dsb sites , would be involved in regulation of the dna - pkcs phosphorylation status and , thus , in regulation of the accessibility of dna ends during the repair process .
it is becoming increasingly clear that dna - pkcs plays a central role during the nhej process .
this enzyme not only captures and tethers the two ends of a broken dna molecule but also regulates the access of modifying enzymes and ligases to the dna termini .
a body of evidence supports a model in which regulation of the accessibility of dna ends is mediated by autophosphorylation of dna - pkcs in trans over the synaptic cleft between the two tethered dna molecules ( fig .
the latest findings that suggest the involvement of atm in the phosphorylation of at least two dna - pkcs residues open a new perspective on the regulation of dna - pkcs in the synaptic complex .
the codependence of dna - pkcs phosphorylation on atm would ensure that at least two signals need to be present before dna - pkcs alters its conformation at the dna ends : ( 1 ) phosphorylation of the atm - responsive sites within the dna - pkcs molecule and ( 2 ) autophosphorylation in trans over the synaptic cleft .
such a mechanism would tightly control dna - pkcs activity , not allowing access to the dna termini until two criteria have been met : a defined dna damage response ( as indicated by activation of the atm kinase ) and a proper alignment of the two dna ends .
until these criteria are met , the dna termini will be protected by the dna - pkcs molecule : a lock with multiple keys . | the dna - dependent protein kinase ( dna - pk ) is one of the central enzymes involved in dna double - strand break ( dsb ) repair .
it facilitates proper alignment of the two ends of the broken dna molecule and coordinates access of other factors to the repair complex .
we discuss the latest findings on dna - pk phosphorylation and offer a working model for the regulation of dna - pk during dsb repair . |
venous air embolism during percutaneous nephrolithotomy ( pcnl ) following air pyelogram or saline irrigation has been reported previously .
we describe a case of suspected air embolism during air pyelogram in a patient undergoing pcnl .
a 32-year - old female patient , weighing 72 kg , with no co - morbidities was posted for pcnl for a 9 mm 16 mm , middle calyx calculus . after recording baseline vitals ( pulse-99/min , bp-132/88 , spo2 - 100 ) balanced general anaesthesia was administered .
induction was carried out with intravenous thiopentone 350 mg and scoline 70 mg were used for intubation .
controlled ventilation was instituted and anaesthesia was maintained with 50% nitrous oxide in oxygen and isoflurane , with intravenous vecuronium as muscle relaxant .
intraoperatively , patient was monitored with ecg , spo2 , etco2 , heart rate , bp measured at 5 min intervals .
the patient was placed in lithotomy position and a ureteric catheter was inserted in left kidney through a cystoscope under fluoroscopic guidance .
after 30 min of the procedure , due to non - visualization of the calculus , another puncture was attempted .
air pyelogram was done by injecting 40 ml of air in increments followed by 10 ml saline for proper identification of the pelvi - calyceal system . within 1 min of injection of air
, there was an abrupt fall in etco2 from 32 mmhg to a sudden 6 mmhg in a span of 10 seconds .
heart rate had elevated from 93/min to 142/min and the bp dropped from 142/90 mmhg to 77/38 mmhg .
endotracheal tube obstruction , circuit disconnection , and anaesthesia machine were checked . since the whole incidence was of transient duration under anesthesia and there was no bronchospam , the airway pressures and tidal volumes remained unchanged .
air embolism was suspected due to sudden drop in etco2 , hypotension , and hypoxemia .
immediately , nitrous oxide was discontinued and manual ventilation with 100% oxygen was carried out .
however , within 3 min , spo2 began to increase , heart rate and etco2 returned to normal values .
anaesthesia was now maintained with 50% air in o2 , isoflurane , and vecuronium . at the end of procedure ,
neuromuscular block was reversed , patient was uneventfully extubated and shifted to post - anaesthesia care unit .
venous air embolism ( vae ) is a potentially life threatening condition that has been reported in several surgical procedures .
cases of vae in patients undergoing retrograde pylography and pcnl have been reported in literature , albeit rarely .
lopez first described that displacement of air occurs due to pyelovenous backflow from renal pelvicaliceal system into renal veins .
injection of large air volume in the confined renal pelvicalyceal system could be potentially hazardous .
air embolism following injection of substantial amount of air occurred in our case as in a report by usha .
air entering the venous system lodges in the pulmonary circulation and causes increased pulmonary vascular resistance , ultimately leading to a fall in the right cardiac output which will cause a decrease in etco2 and spo2 .
the factors that influence the morbidity and mortality of vae include volume of air entrained , rate of air entrainment , position of the patient during the event , and the cardiac status of the patient .
prone position is detrimental as it produces significant gravitational gradient between right heart and the renal pelvis .
the application of end tidal carbon dioxide monitoring during general anaesthesia is a routine practice and could provide a sensitive and specific method to detect vae . nowadays , in surgeries where air embolism is likely , invasive monitoring such as intra - arterial blood pressure monitoring , trans - oesophageal echocardiography , precordial doppler
a right atrial catheter is inserted for diagnostic and therapeutic purpose . in short procedures , like pcnl where air embolism is a rare complication ,
hence , detection of air embolism under general anaesthesia is difficult and requires a high index of suspicion and prompt treatment . in our case
as soon as the etco2 and spo2 dropped and hemodynamic instability ensued , air embolism as a probable cause was considered .
we ruled out other possibilities like cardiac arrhythmia , pneumothorax , displacement of the endotracheal tube , ventilatory or mechanical failure can simulate air embolism .
high concentration of oxygen produces a gradient which could displace nitrous oxide from the air bubble .
rapid intravenous fluid therapy instituted as low central venous pressure and hypotension would favor further air entrainment by increasing the pressure gradient between open veins and the right atrium .
after patient was ensured to be hemodynamically stable surgery was allowed to be continued ; however , nitrous oxide was not reinstituted to prevent increase in the size of any residual bubbles .
usha reported a similar case of air embolism during pcnl , however their patient developed asystole and was required to be turned supine and resuscitated .
a similar case of intra - operative air embolism during pcnl resulting in paradoxical air embolism was described by droghetti et al .
, air embolism should be kept in mind following air pyelogram during pcnl surgery . this may be avoided or minimized by using smaller amounts of air or other alternate gas like co2 .
vigilant monitoring with available monitors , particularly end tidal co2 and prompt treatment could result in favorable outcome following air embolism . | venous air embolism during percutaneous nephrolithotomy ( pcnl ) following air pyelogram or saline irrigation has been occasionally reported .
we present a case of suspected venous air embolism during air pyelogram in a patient undergoing pcnl .
the clinical diagnosis of air embolism was made by fall in end tidal carbon dioxide , blood pressure and oxygen saturation and was conservatively managed .
early diagnosis with rapid resuscitation is the key to management of a patient with air embolism . |
tetracyclines are a broad - spectrum antibiotics family active against a wide range of microorganisms including gram - positive and gram - negative bacteria , chlamydiae , mycoplasmas , rickettsiae , and protozoan parasites [ 1 , 2 ] .
thereafter , other natural tetracyclines were isolated , including tetracycline , for which the family of molecules is named . since then
, the manipulation of natural antibiotics and the laboratory synthesis of novel compounds have enriched the tetracycline family . compared with the original tetracycline
, the synthetics , including minocycline and doxycycline , show a better pharmacokinetic profile than the first - generation tetracyclines when used orally , being rapidly and completely absorbed , even in elderly populations , with a longer half - life and excellent tissue penetration , with almost complete bioavailability [ 4 , 5 ] .
in addition to their well - characterized antibiotic effects , criticized in recent decades for the emergence of bacterial resistance , extensive research on tetracyclines has revealed a range of vastly important pharmacological properties focused on the regulatory influence on the immune system and inflammatory pathway .
the nonantibiotic anti - inflammatory effects of tetracyclines are under investigation for the treatment of multiple diseases .
their efficacy has already been reported in rheumatoid arthritis , corneal inflammation , gingivitis , osteoarthritic cartilage , allergen - induced inflammation , and a lot of other conditions like cardiovascular , neurological , and skin disorders [ 1 , 7 , 8 ] .
the wide spectrum of anti - inflammatory effects ascribed to tetracyclines is probably due to their ability to interfere with the synthesis or activity of several mediators of inflammation .
in addition , tetracyclines inhibit metalloproteinases ( mmps ) and suppress hydrolases such as -amylases and phospholipase a2 ( key enzyme in the biosynthesis of inflammatory mediators such as the prostaglandins ( pge ) ) [ 79 ] .
furthermore , tetracyclines also scavenge reactive oxygen species and thereby prevent or reduce pathological tissue destruction . another important effect , significantly contributing to the overall immune - regulatory activity of tetracyclines , is the interference with cytokine production from neutrophils and macrophages during inflammatory conditions .
tetracyclines suppress cytokines , such as tumor necrosis factor - alpha ( tnf- ) , interleukin- ( il- ) 1 , and il-6 , involved in inflammatory disorders as seborrheic dermatitis , psoriasis , acne , and rosacea [ 710 ] .
for some of these diseases , principally , acne and rosacea , high doses of doxycycline ( from 100 to 200 mg / die ) are often administered , although they are frequently considered responsible for development of bacterial resistances and endogenous flora alterations . concerning acne and rosacea treatment ,
quantitative bacteriologic studies demonstrate greater in vivo antimicrobial activity of minocycline compared with doxycycline and tetracycline against propionibacterium acnes [ 11 , 12 ] .
ishikawa et al . ( 2009 ) reported that minocycline reduces the protease - activated receptor 2-mediated production of il-8 and thus attenuates the proinflammatory process in epidermal keratinocytes .
however , although minocycline is recommended for the aforementioned skin disorders , current therapy has moved towards the use of doxycycline , which has a similar efficacy for the treatment of these conditions with a reduced incidence of adverse effects , such as drug hypersensitivity syndrome , hyperpigmentation , and dizziness , frequently associated with minocycline therapy [ 8 , 14 , 15 ] . in recent clinical trials ,
lower doxycycline dose ( 40 mg / day ) seems to be connected with the improvement of clinical manifestations in some dermatitis .
low dose , administered once daily , appears to be effective and safe for the treatment of moderate to severe papulopustular rosacea , without bacterial flora alteration [ 10 , 16 ] .
the reason why this dose of doxycycline may produce this result is probably related to the specific involvement of lower doses of doxycycline in inflammatory mechanisms . for this reason , aim of our study was to investigate , in vitro , if low doxycycline doses could be able to downregulate some of the cytokines involved in the pathogenesis of inflammatory skin diseases .
immortalized human keratinocytes ( hacat ) were grown in dulbecco 's modified eagle 's medium ( dmem , gibco , grand island , ny ) containing 10% fetal bovine serum ( fbs , gibco , grand island , ny ) , 2 mm l - glutamine ( gibco , grand island , ny ) , and antibiotics ( 100 iu / ml penicillin g and 100 g / ml streptomycin , gibco , grand island , ny ) .
hacat cells were seeded onto 6-well culture plates in 2 ml of fresh culture medium until they reached a confluence of about 70% .
after incubation for 1 day at 37c in 5% co2 , cells were washed with phosphate - buffered saline ( pbs , gibco , grand island , ny ) , covered with dmem without serum , and stimulated with 25 g / ml of bacterial endotoxin - lipopolysaccharide ( lps ) a ( sigma - aldrich , oakville , on , canada ) in absence or presence of doxycycline ( 0.3 mg/l ; 0.6 mg/l ; 1.5 mg/l ; 3 mg/l ) ( sigma - aldrich , oakville , on , canada ) .
these selected in vitro doses try to reproduce doxycycline plasma levels theoretically obtained after in vivo administration of 20 , 40 , 100 , and 200 mg / day , respectively .
literature data indicate that the high doxycycline doses ( 100200 mg / day ) used to treat rosacea correspond to a plasma concentration of 1.53 mg/l , 2 - 3 hours after oral intake . from that
, we extrapolated the theoretical doxycycline blood concentrations of 0.3 and 0.6 mg/l obtained 3 hours after low doses ( 2040 mg ) oral assumption .
to evaluate the protective or restoring doxycycline properties on lps - stimulated hacat cells , three experimental settings were used , differing in the timing of doxycycline treatment in respect to the insult induced by lps : pretreatment , concomitant , and posttreatment . for the pretreatment setting
, cells were preincubated with doxycycline for 24 hours before receiving lps ; thereafter , 24 hours later , cells were harvested and mrna extraction was performed : this was 48 hours after doxycycline incubation and 24 hours after lps stimulation . in the concomitant
setting , lps and doxycycline were simultaneously added to the cells and mrna was extracted 24 hours later .
lastly , for the posttreatment assessment , cells were stimulated with lps for 24 hours and thereafter doxycycline was added ; cells were incubated for further 24 hours and rna extraction was performed at 48 hours ( figure 1 ) .
rna was extracted ( rneasy mini protocol qiagen , valencia , ca ) from hacat cells and cdna was prepared according to the manufacturer 's instructions .
qrt - pcr ( transcriptor high fidelity cdna synthesis , roche , indianapolis , in ) was used to analyze il-8 , tnf- , and il-6 gene expression .
pcr primers were designed based on published sequences , and their specificity was verified with blast alignment search .
the amount of mrna for a given gene in each sample was normalized to the amount of mrna of 18s reference gene in the same sample .
il-8 , tnf- , and il-6 protein levels were measured in supernatants from hacat cells stimulated or not with lps ( 25 g / ml ) at 24 and 48 h by single enzyme - linked immunosorbent assay ( elisa ) , using commercially available kits ( r&d systems , minneapolis , mn , usa ) , according to the manufacturer 's instructions .
all statistical analyses were performed using graphpad prism 4.0 ( graphpad software inc . , la jolla , ca ) .
data that passed the normality test were analysed with two - tailed t - test .
data are expressed as means sd of three independent experiments , each performed in triplicate .
doxycycline , at the four tested concentrations , did not exert any toxic effect toward hacat cells and after either 24 or 48 hours cellular viability results were similar to controls ( data not shown ) .
lps , as expected , considerably reduced cell viability of 20 , 28 , and 36% in pretreatment , concomitant , and posttreatment settings , respectively .
doxycycline was unable to antagonize this toxic effect ( figure 2 ) ; nevertheless cell viability after lps was considered sufficient to perform our experiments and to permit a correct analysis , as reported in previous investigations .
lps enhanced mrna levels of il-8 , tnf- , and il-6 in the three experimental settings ( figures 3 , 4 , and 5 ) .
these data were confirmed by the assessment of proteins in supernatants from hacat cells stimulated with lps ( 25 g / ml ) at 24 and 48 h ( figure 6 ) .
low doxycycline doses were more effective than high doses in modulating gene expression of lps - induced proinflammatory cytokines ( il-8 , tnf- , and il-6 ) when added before or after lps stimulation ( figures 35 , panels ( a ) and ( c ) ) .
when lps and doxycycline were simultaneously added to cell cultures high doses were more effective ( figures 35 , panels ( b ) ) .
in particular , il-8 increments , induced by lps , showed a significant reduction ( 68.7 and 56.7% ) in the pretreatment setting with low doxycycline doses ( 0.3 and 0.6 mg/l ) ( figure 3(a ) ) , as well as in posttreatment setting ( 64.3 and 66% ) ( figure 3(c ) ) . in a reduced manner ,
high doses were also able to decrease il-8 levels in pretreatment setting ( figure 3(a ) ) and slightly significant differences were observed in posttreatment setting ( figure 3(c ) ) . in regard to tnf- , even if lps induced a modest increase in the pretreatment setting , low doxycycline doses strongly downregulated its expression ( 86.4 and 86.7% ) , whereas high doses were able to induce a more moderate reduction ( figure 4(a ) ) .
also in the posttreatment setting , tnf- expression was better reduced by low doses ( 87.6 and 86.4% ) whereas , of the two high doses , statistically significant effect was achieved only by doxycycline 3 mg/l ( figure 4(c ) ) .
il-6 increments were reduced either by low or high doses in pretreatment setting ( 80.2 and 81.6% ) ( figure 5(a ) ) , whereas in the posttreatment setting significant differences were observed only for doxycycline 0.3 and 0.6 mg/l ( figure 5(c ) ) . in the concomitant treatment ,
only high doxycycline concentrations were able to determine a statistically significant reduction in il-8 , tnf- , and il-6 expression ( figures 3(b ) , 4(b ) , and 5(b ) ) , whereas low doses could not reduce inflammation but , apparently , enhanced the lps - induced ils increase .
the rationale of the present study comes from the observation that the traditional antimicrobial doses of doxycycline can exert selection pressure , altering normal commensal microflora and increasing the risk of bacterial resistance [ 69 , 20 ] .
side effect has not been detected , even during long - term treatment , using low doses of doxycycline ( 2040 mg / day ) , able to exert their anti - inflammatory activities without altering bacteria susceptibility to antibiotics [ 16 , 20 , 21 ] .
herein , using immortalized keratinocytes stimulated with lps , we provided evidence that both low and high doxycycline doses were able to modulate the expression of inflammatory mediators , in accordance with previous studies illustrating the capability of both anti - inflammatory and antimicrobial doses to be equally effective in rosacea treatment .
we have chosen hacat as cellular model because these cells are widely used in mechanistic and pharmacological studies and because they represent an easy way to handle substitutes for primary human keratinocytes .
recently , keratinocytes appear no longer as passive bystanders in the pathobiology of rosacea but actively contribute to inflammation and fibrosis specially by releasing proinflammatory cytokines , chemokines , growth factors , mmps , and proteases [ 2224 ] . therefore keratinocytes are not only primary sensors of stressful conditions but also major players of the extremely complex immune response in the skin .
however , the exact role of keratinocytes in the context of rosacea still needs to be explored . in response to various external stimuli and/or to endogenous proinflammatory cytokines ,
epidermal keratinocytes release chemokines , such as il-8 , which recruit neutrophils and lymphocytes into the skin and exacerbate inflammation .
several studies demonstrate that tetracyclines exert a range of nonantibiotic activities achieved at doses below the level needed for antibacterial ones , resulting in very interesting molecules for the treatment of a variety of noninfectious diseases , including rosacea . at present , doxycycline is the only tetracycline for which a dosage separation between antibiotic and anti - inflammatory effects has been established .
our findings regarding doxycycline capability to reduce cytokine production in the lps - induced model inflammation agree with those of yoshimura et al . and allon et al . , who demonstrated the beneficial effects of low doxycycline doses in several inflammatory diseases [ 2931 ] .
interestingly , from our results , lower doxycycline doses were more effective than the higher ones , when added before or after lps stimulation , but not in case of concomitant treatment when lps and doxycycline were added simultaneously to cell cultures .
particularly , in our experiments , il-8 increments showed a significant reduction in case of pretreatment with doxycycline 0.3 and 0.6 mg/l but also in case of posttreatment .
otherwise , high doxycycline doses ( 1.5 and 3 mg/l ) were able to reduce il-8 levels in the pretreatment setting , whereas no significant differences were observed in posttreatment setting .
il-8 is a potent neutrophil chemoattractant and activating factor produced by a variety of cells , including keratinocytes , and its production is also augmented by tnf- .
son et al . found that endogenous il-8 mrna expression was reduced by doxycycline treatment in a dose - dependent manner in pancreatic cancer cells ( panc-1 ) and that tnf--induced il-8 mrna expression was effectively blocked by 48 h of doxycycline pretreatment .
further surveys have shown that low doxycycline doses downregulate proinflammatory cytokines , including tnf- and il-1 in multiple models [ 79 ] . according to these authors , it is unclear whether the mechanism for this effect is an indirect result of other anti - inflammatory actions or whether tetracyclines have a more direct effect on cytokines release .
they suggest the existence of a posttranslational effect since levels of secreted cytokines were decreased , whereas there was no decrease in cytokine mrna .
this is partially in contrast with our results : we proved that doxycycline significantly modulates tnf- gene expression ( figure 4 ) but also il-8 and il-6 ( figures 3 and 5 ) , and this would probably correlate with proteins modulation .
in fact , the increased gene expression found in hacat cell after lps is confirmed in our study by elisa ( figure 6 ) . with regard to il-6 expression , we found that both low and high doses significantly affected the increase induced by lps in doxycycline pretreatment , whereas significant differences were observed only for 0.3 and 0.6 mg/l concentrations in the posttreatment setting .
our data agree with ikeda - dantsuji et al . who reported that the production of il-6 was suppressed by the minimum inhibitory concentrations of doxycycline in trachomatis - infected human fibroblast - like synovial cells ( hfls ) or hela 229 cells immediately or early after infection .
il-6 is a multifunctional cytokine , produced by monocytes and keratinocytes upon inflammatory stimulation , such as exposure to uv radiation and bacterial endotoxin , whose main functions include activation of lymphocytes and induction of an acute phase response .
although low doses of doxycycline were more effective in the pretreatment and posttreatment settings , in the concomitant treatment analysis only the higher concentrations of doxycycline were able to produce a statistically significant reduction of il-8 , tnf- , and il-6 expression , whereas low doxycycline doses apparently enhanced the lps - induced ils increase .
this effect could be explained by the cellular responses to the stress caused by the concomitant addition of two substances .
indeed , these cells have been weakened by culture with serum - free medium , so that it seemed that they were not able to distinguish between lps and low dose doxycycline stimuli , sensing both of them as proinflammatory triggers .
pastore et al . , in a study on environmental and endogenous stresses to the skin as causative of cancer , demonstrated that plant polyphenols ( pps ) , known for their antioxidant activity , used in combination with uva or lps stimuli induced strong increase of stress responses in hacat .
therefore , molecular responses of hacat to a combination of lps and pps showed synergism between the two factors in upregulation of proinflammatory cytokines gene and protein expression .
probably , low doxycycline doses are more effective to prevent inflammation ( pretreatment setting ) or to reduce it if the process is already in progress ( posttreatment ) , because they are rapidly absorbed and have a prolonged half - life , with potential preventive or antagonistic effect toward the damaging stimuli ( lps in our case ) .
inflammatory process usually involves three phases associated with different pattern : acute inflammation , immune response , and chronic inflammation .
the acute inflammation characterizes the initial response to tissue injury and is related to the release of autacoids as histamine , serotonin , bradykinin , prostaglandins , and leukotrienes .
acute inflammation presents a relatively short duration and may be followed by an immune response characterized by the activation of immune cells and by chronic inflammation .
thus , the mechanisms involved in the cellular and molecular pattern of acute and chronic inflammations are different .
although there are some reports on the efficacy of second generation tetracyclines on central inflammatory conditions [ 4042 ] , there are few reports regarding their anti - inflammatory effects in peripheral acute processes .
our study provides in vitro evidence that both low and high doxycycline doses are able to directly modulate the expression of inflammatory mediators , in accordance with previous in vivo studies , highlighting the better efficacy of low doses in multiple settings .
thereafter , we suggest that low doxycycline doses could be safely used in case of chronic or acute inflammatory skin disease in which the inflammatory process , either constantly in progress or periodically recurring , has to be prevented or controlled . | doxycycline is used to treat infective diseases because of its broadspectrum efficacy .
high dose administration ( 100 or 200 mg / day ) is often responsible for development of bacterial resistances and endogenous flora alterations , whereas low doses ( 2040 mg / day ) do not alter bacteria susceptibility to antibiotics and exert anti - inflammatory activities . in this study
, we wanted to assess the efficacy of both low and high doxycycline doses in modulating il-8 , tnf- , and il-6 gene expression in hacat cells stimulated with lps .
three experimental settings were used , differing in the timing of doxycycline treatment in respect to the insult induced by lps : pretreatment , concomitant , and posttreatment .
low doses were more effective than high doses in modulating gene expression of lps - induced proinflammatory cytokines ( il-8 , tnf- , and il-6 ) , when added before ( pretreatment ) or after ( posttreatment ) lps stimulation .
this effect was not appreciated when lps and doxycycline were simultaneously added to cell cultures : in this case high doses were more effective . in conclusion
, our in vitro study suggests that low doxycycline doses could be safely used in chronic or acute skin diseases in which the inflammatory process , either constantly in progress or periodically recurring , has to be prevented or controlled . |
to date , there is a wide literature focusing on the attachment theory1 as a possible framework for understanding anorexia nervosa origin , course and treatment response.24 despite the increasing evidences of dysfunctional family relations and insecure style of attachment in eating - disordered patients,57 literature data are contradictory and limited by a wide variety of methodological approaches.8 to summarize the findings regarding attachment in eating disorders ( eds ) , it is worth noticing that insecure attachment ( dismissing or preoccupied types ) is frequent among patients with eds : in particular , avoidant attachment tends to be more represented in patients with restricting anorexia , while patients with bulimia show a higher prevalence of attachment preoccupation.8 on the whole , patients with eds often report problems with childhood family relationships,8 but the precise nature of the problems and the mechanism underlying their connection with eds are rather unclear , since findings from scientific literature appear to be still inconclusive.3,8,9 literature exploring the connection between eds and early parent
child relations include studies based on the attachment theory as well as on the parental bonding construct.8,9 both of the constructs provide information about the family relationships , as subjectively perceived by an individual.10 however , if the attachment construct refers prevalently to how the child responds to parental behaviors , the parental bonding construct instead addresses mainly the representation of the parents contribution to the parent child relation.11 several studies dealt with the parental bonding , specifically investigating the two fundamental dimensions of care and protection , and providing often conflicting results due to the studied population ( clinical or nonclinical groups of different ages ) , and to the ed diagnostic subtype . for what concerns adult samples
only , the majority of the findings points to a lower parental care perceived by patients with eds , along with a sort of overprotection.12 in their systematic review , however , tetley et al12 suggested that this characteristic might not be specific to patients with eds , as they do not differ in the care and protection levels from patients with other psychiatric disorders .
moreover , the studies have to date failed in establishing a potential distinction among different ed diagnoses as regards the perceived parental bonding . only very few studies13,14 have specifically investigated parental bonding in adolescents with anorexia , although adolescence is very often the moment of the anorexic onset and at the same time the more fruitful life stage to treat it.15,16 it is worth noticing that , due to the specificity of the adolescent period , the results obtained in the adult sample are not always replicated unchanged in the adolescent samples .
in fact , somehow surprisingly , both studies available13,14 found the adolescents with restricting - type anorexia to perceive an optimal parental bonding with both mothers and fathers . unlike the binge - eating / purging - type patients,14
the former even perceive a better care and a lower control on the parents part , compared to the adolescents of the general population included in the control groups . for what concerns the first study by russell et al,13 the explanation of the striking result may lay in the different treatment ( and in the different treatment stage ) already received by most of the patients included in the study .
not only the daughters in treatment but also the parents , receiving parental counseling , are plausibly making an effort to change their previous attitudes within the family relations , striving to give a positive image .
another possible explanation concerns the tendency to idealization and defensive avoidance , usually exhibited by the patients with restrictive conducts , which may use dismissing attachment strategies in order to control the negative feelings.3,8,14 furthermore , there has been little research specifically into the parents own attachment patterns and perceived bonding , and whether there might be intergenerational transmission of these patterns remains still unclear .
ward et al7 hypothesized an intergenerational transmission of attachment patterns from the mothers to the daughters with anorexia .
although patients with anorexia and their mothers commonly showed a dismissive attachment style , the authors did not find an association between mothers and daughters attachment.7 nevertheless , the study suggested that mothers of women with anorexia displayed a high rate of unresolved losses , which implies a lower capacity in the reflective functioning and emotional regulation on the mothers part , which may be transmitted or learned from mother to daughter .
this kind of maternal psychological functioning might therefore be considered as a risk factor for the daughters development of anorexia nervosa.3,7 in line with those results , specificities on the paternal as well as on the maternal parental bonding toward their own parents were found by the only study17 conducted exclusively on adult patients , which has at least in part addressed the intergenerational transmission of parental bonding styles , administering the parental bonding instrument ( pbi ) questionnaire to parents of patients with eds .
literature is , however , completely lacking in data on the intergenerational transmission of parental bonding and attachment representations in families of adolescent patients with eds , and in particular with anorexia nervosa , and hence , the present study was conducted .
in particular , the first aim of the study was to investigate the perceived parental bonding , as described by the pbi , in a group of adolescents newly diagnosed with restricting - type anorexia , comparing them to healthy controls .
the second and main aim of the study was to broaden the limited knowledge on parental bonding in adolescents with anorexia and in their parents and to evaluate whether the families of adolescents with anorexia exhibit specific patterns of parental bonding , in order to better understand if these specific patterns can be transmitted from generation to generation . to achieve these goals , a latent class analysis ( lca ) approach
was adopted to identify specific pattern of parental bonding transmission ( separately for the maternal and the paternal bonding ) comparing the families of patients with anorexia with families belonging to the general population .
the perceived parental bonding was evaluated for what concerns both the mother and the father of the patients . moreover , parental perceptions of their family of origin were evaluated as well , in order to adopt a family - level approach .
they belonged to the 26 families of patients with anorexia and the 30 families of the control group .
the adolescents had been consecutively admitted as inpatients to the child and adolescent neuropsychiatry unit , national neurological institute irccs c mondino , university of pavia , italy , and the recruitment lasted 5 months .
selection for the anorexia group was based on the following inclusion criteria : new diagnosis of anorexia nervosa , restricting subtype in accordance with the criteria of the diagnostic and statistical manual of mental disorders , 4th edition , text revision ( dsm - iv - tr);18 age between 12 and 18 years ; and current body mass index ( bmi ) below the tenth percentile per age and sex .
exclusion criteria instead were another major diagnosis ( autism , schizophrenia and learning disabilities ) and insufficient understanding of the italian language .
adolescents for the control group were selected according to the following inclusion criteria : normal weight and age between 12 and 18 years .
exclusion criteria for the families of the control group were presence of adolescent s symptoms related to food ( either restrictive or purging or binge - eating conducts ) or other psychopathological symptoms , and insufficient understanding of the italian language . the mean age of the 26 adolescent patients with anorexia ( 23 females and 3 males ) was 14.62 years ( standard deviation=2.00 ; range 1218 years ) , and their mean current bmi was 15.55 ( standard deviation=1.34 ) .
the control group was made up of 30 healthy subjects ( 27 females and three males ) , and the mean age of the adolescents was 14.30 years ( standard deviation=2.07 ; range 1218 years ) .
child and adolescent psychiatrists and clinical psychologists , not directly involved in the research project , recruited the patients and their parents and obtained their informed consent .
a child neuropsychiatrist with special interest and clinical experience in adolescent anorexia evaluated all patients and collected a comprehensive medical and family history .
patients were diagnosed according to the dsm - iv - tr criteria for restricting - type anorexia nervosa.18 the diagnosis of potential comorbid disorders was supported by the kiddie - sads semi - structured interview.19 the control group was composed of parents and offspring , randomly recruited among the adolescents attending two local secondary schools .
the agreement of the educational authorities was obtained , and the parents of the participating adolescents were required to give their informed consent to the study .
to exclude a history of ed and the presence of psychopathological and behavioral disorders , the adolescents of the control group and their parents separately underwent a detailed anamnestic interview , during which a trained child and adolescent psychiatrist also administered the eating disorder inventory-220 to the adolescents . to evaluate parental rearing styles , all the participating adolescents and their parents
were administered the pbi questionnaire.11 in particular , the adolescents with anorexia and their parents were requested to answer the questionnaire during their first week of hospitalization .
all parents and patients signed an informed consent , reviewed by the institutional review board of the c mondino institute of neurology of pavia , which approved the study .
the pbi is a 25-item self - report measure of two fundamental dimensions of parental bonding , care and overprotection , concerning the mother and the father .
twelve care items reflect the dimension of affection and nurturing versus emotional rejection and neglect .
thirteen overprotection items assess the dimension of rigid control versus the fostering of autonomy . according to parker et al s instructions,11 the pbi items
were rated on a likert scale , ranging from 1 ( very likely ) to 4 ( very unlikely ) . on the basis of the care and overprotection values emerging from the application of the questionnaire , four parenting styles
have been defined : optimal bonding ( high care and low overprotection ) , absent or weak bonding ( low care and overprotection ) , affectionless control ( high overprotection and low care ) and affectionate constraint ( high care and high overprotection ) ( figure 1 ) .
good psychometric properties of the scale , with cronbach s ranging from 0.74 to 0.95 , have also been reported in literature.11 as a first approach to the data , in order to examine at a manifest level the pbi responses , before applying the lca , the parental care and overprotection variables were analyzed by comparing the means of the two groups of families , using student s t - test .
the lca approach21 was chosen to identify a latent categorical variable , namely to identify a hypothetical construct , accounting for the covariance between the observed variables .
the lca assumes the latent variable to be categorical and composed by a number of classes , which describe the presence or absence of the characteristics of the latent variable .
each class should represent a profile in relation to specific variables that are in this study : family roles , care and overprotection , typical of families of adolescents with anorexia and families of the control group .
one latent variable with two classes representing the two family groups , families of adolescents with anorexia and families of the control group , was hypothesized in this context .
the two latent class structures were supposed to be different in relation to the maternal and paternal bonding .
however , given the explorative nature of the modeling approach used herein , no specific hypothesis about how the paternal bonding structure and the maternal bonding structure would differ was put forward . the variables ( manifest variables ) introduced in the lca model were the group ( anorexia and control ) , the family role ( adolescent , mother and father ) , the pbi variable care ( two levels : low care and high care ) and the pbi variable overprotection ( two levels : low overprotection and high overprotection ) .
the lca approach has been extensively used in the psychological and medical literature to model the relations among variables in order to identify the underlying structure characterized by one or more latent categorical variables and draw a profile of people falling into one class or another.22,23 the model fitting the data were assessed by looking at the akaike information criterion ( aic ) and the bayesian information criterion ( bic ) .
the model is considered to have a good fit if both statistics have a probability higher than p=0.05 .
they belonged to the 26 families of patients with anorexia and the 30 families of the control group .
the adolescents had been consecutively admitted as inpatients to the child and adolescent neuropsychiatry unit , national neurological institute irccs c mondino , university of pavia , italy , and the recruitment lasted 5 months .
selection for the anorexia group was based on the following inclusion criteria : new diagnosis of anorexia nervosa , restricting subtype in accordance with the criteria of the diagnostic and statistical manual of mental disorders , 4th edition , text revision ( dsm - iv - tr);18 age between 12 and 18 years ; and current body mass index ( bmi ) below the tenth percentile per age and sex .
exclusion criteria instead were another major diagnosis ( autism , schizophrenia and learning disabilities ) and insufficient understanding of the italian language .
adolescents for the control group were selected according to the following inclusion criteria : normal weight and age between 12 and 18 years .
exclusion criteria for the families of the control group were presence of adolescent s symptoms related to food ( either restrictive or purging or binge - eating conducts ) or other psychopathological symptoms , and insufficient understanding of the italian language . the mean age of the 26 adolescent patients with anorexia ( 23 females and 3 males ) was 14.62 years ( standard deviation=2.00 ; range 1218 years ) , and their mean current bmi was 15.55 ( standard deviation=1.34 ) .
the control group was made up of 30 healthy subjects ( 27 females and three males ) , and the mean age of the adolescents was 14.30 years ( standard deviation=2.07 ; range 1218 years ) .
child and adolescent psychiatrists and clinical psychologists , not directly involved in the research project , recruited the patients and their parents and obtained their informed consent .
a child neuropsychiatrist with special interest and clinical experience in adolescent anorexia evaluated all patients and collected a comprehensive medical and family history .
patients were diagnosed according to the dsm - iv - tr criteria for restricting - type anorexia nervosa.18 the diagnosis of potential comorbid disorders was supported by the kiddie - sads semi - structured interview.19 the control group was composed of parents and offspring , randomly recruited among the adolescents attending two local secondary schools .
the agreement of the educational authorities was obtained , and the parents of the participating adolescents were required to give their informed consent to the study .
to exclude a history of ed and the presence of psychopathological and behavioral disorders , the adolescents of the control group and their parents separately underwent a detailed anamnestic interview , during which a trained child and adolescent psychiatrist also administered the eating disorder inventory-220 to the adolescents . to evaluate parental rearing styles , all the participating adolescents and their parents
were administered the pbi questionnaire.11 in particular , the adolescents with anorexia and their parents were requested to answer the questionnaire during their first week of hospitalization .
all parents and patients signed an informed consent , reviewed by the institutional review board of the c mondino institute of neurology of pavia , which approved the study .
the pbi is a 25-item self - report measure of two fundamental dimensions of parental bonding , care and overprotection , concerning the mother and the father .
twelve care items reflect the dimension of affection and nurturing versus emotional rejection and neglect .
thirteen overprotection items assess the dimension of rigid control versus the fostering of autonomy . according to parker et al s instructions,11 the pbi items
were rated on a likert scale , ranging from 1 ( very likely ) to 4 ( very unlikely ) . on the basis of the care and overprotection values emerging from the application of the questionnaire , four parenting styles
have been defined : optimal bonding ( high care and low overprotection ) , absent or weak bonding ( low care and overprotection ) , affectionless control ( high overprotection and low care ) and affectionate constraint ( high care and high overprotection ) ( figure 1 ) .
good psychometric properties of the scale , with cronbach s ranging from 0.74 to 0.95 , have also been reported in literature.11
as a first approach to the data , in order to examine at a manifest level the pbi responses , before applying the lca , the parental care and overprotection variables were analyzed by comparing the means of the two groups of families , using student s t - test .
the lca approach21 was chosen to identify a latent categorical variable , namely to identify a hypothetical construct , accounting for the covariance between the observed variables .
the lca assumes the latent variable to be categorical and composed by a number of classes , which describe the presence or absence of the characteristics of the latent variable .
each class should represent a profile in relation to specific variables that are in this study : family roles , care and overprotection , typical of families of adolescents with anorexia and families of the control group .
one latent variable with two classes representing the two family groups , families of adolescents with anorexia and families of the control group , was hypothesized in this context .
the two latent class structures were supposed to be different in relation to the maternal and paternal bonding .
however , given the explorative nature of the modeling approach used herein , no specific hypothesis about how the paternal bonding structure and the maternal bonding structure would differ was put forward . the variables ( manifest variables ) introduced in the lca model were the group ( anorexia and control ) , the family role ( adolescent , mother and father ) , the pbi variable care ( two levels : low care and high care ) and the pbi variable overprotection ( two levels : low overprotection and high overprotection ) . the lca approach has been extensively used in the psychological and medical literature to model the relations among variables in order to identify the underlying structure characterized by one or more latent categorical variables and draw a profile of people falling into one class or another.22,23 the model fitting the data were assessed by looking at the akaike information criterion ( aic ) and the bayesian information criterion ( bic ) .
the model is considered to have a good fit if both statistics have a probability higher than p=0.05 .
with the aim of exploring the perceived parental bonding , measured by the pbi , in a group of adolescents newly diagnosed with restricting - type anorexia , and in their families , patients and parents were compared with adolescents and parents belonging to families from the general population . in table 1 , pbi care and overprotection scales scores are reported .
the adolescents presented similar scores , whereas significant differences emerged between parents of adolescents with anorexia and parents of healthy controls ( table 1 ) .
the lca identified the two - latent - class models as hypothesized both for paternal and maternal bonding .
the indices fitting the data were chi square = 9.90 , df=6 , p=0.13 , l2=9.99 , df=6 , p=0.13 , aic ( l2 ) = -2.01 and bic ( l2 ) = -2.75 for paternal bonding , and chi square = 11.73 , df=6 , p=0.06 , l2=11.11 , df=6 , p=0.16 , aic ( l2 ) = -1.11 and bic ( l2 ) = -17.63 for maternal bonding .
once the latent structure was identified , the probability parameter estimates for each latent class revealed their distinctive characteristics ( table 2 ) .
for both paternal and maternal bonding , in general , classes 1 and 2 presented rather similar probabilities , in the range 0.450.55 , but each class evidenced different structures , which allowed the interpretation of the data .
substantial probability values used for the latent class interpretation are evidenced in bold in table 2 .
maternal bonding is characterized as follows : class 1 is represented by the families of patients with anorexia ( 0.52 ) , and the maternal bonding comes through the father s transmission line ( 0.39 ) and is characterized by low care ( 0.75 ) and high overprotection ( 64 ) .
class 2 is represented by the families of the control group ( 0.59 ) , and in this case , the maternal bonding comes through the mother s transmission line ( 0.38 ) and is characterized by high care ( 0.54 ) and low overprotection ( 0.71 ) .
similarly , as far as paternal bonding is concerned , class 1 is represented by the families of patients with anorexia ( 0.61 ) .
in particular , the paternal bonding comes through the father s transmission line ( 0.44 ) and is characterized by low care ( 0.81 ) and low overprotection ( 0.59 ) .
class 2 instead is represented by the control group ( 0.71 ) , where the paternal bonding comes through her mother s transmission line and is characterized by high care ( 0.79 ) and low overprotection ( 0.58 ) .
considering now the two groups , families of patients with anorexia versus control families , it is possible to evidence differences and analogies between them . for families of adolescents with anorexia ( class 1 ) ,
both maternal ( 0.52 ) and paternal ( 0.61 ) bonding comes through the father s transmission line ( 0.39 and 0.44 , respectively , for maternal and paternal bonding ) .
in fact , the adolescents father bonding is characterized by specificities concerning the relationship recalled both with his father and his mother .
however , the kind of transmission is not the same ; that is , maternal bonding is characterized by low care ( 0.75 ) and high overprotection ( 0.64 ) , whereas paternal bonding is characterized by low care ( 0.81 ) and low overprotection ( 0.59 ) .
for control families ( class 2 ) , both maternal ( 0.59 ) and paternal ( 0.71 ) bonding comes through the mother s transmission line ( 0.38 and 0.46 , respectively , for maternal and paternal bonding ) . the parental bonding recalled by the control adolescents mother
maternal and paternal bonding are characterized in fact by high care ( 0.54 and 0.79 , respectively ) and low overprotection ( 0.71 and 0.58 , respectively ) .
with the aim of exploring the perceived parental bonding , measured by the pbi , in a group of adolescents newly diagnosed with restricting - type anorexia , and in their families , patients and parents were compared with adolescents and parents belonging to families from the general population . in table 1 , pbi care and overprotection scales scores are reported .
the adolescents presented similar scores , whereas significant differences emerged between parents of adolescents with anorexia and parents of healthy controls ( table 1 ) .
the lca identified the two - latent - class models as hypothesized both for paternal and maternal bonding .
the indices fitting the data were chi square = 9.90 , df=6 , p=0.13 , l2=9.99 , df=6 , p=0.13 , aic ( l2 ) = -2.01 and bic ( l2 ) = -2.75 for paternal bonding , and chi square = 11.73 , df=6 , p=0.06 , l2=11.11 , df=6 , p=0.16 , aic ( l2 ) = -1.11 and bic ( l2 ) = -17.63 for maternal bonding .
once the latent structure was identified , the probability parameter estimates for each latent class revealed their distinctive characteristics ( table 2 ) .
for both paternal and maternal bonding , in general , classes 1 and 2 presented rather similar probabilities , in the range 0.450.55 , but each class evidenced different structures , which allowed the interpretation of the data .
substantial probability values used for the latent class interpretation are evidenced in bold in table 2 .
maternal bonding is characterized as follows : class 1 is represented by the families of patients with anorexia ( 0.52 ) , and the maternal bonding comes through the father s transmission line ( 0.39 ) and is characterized by low care ( 0.75 ) and high overprotection ( 64 ) .
class 2 is represented by the families of the control group ( 0.59 ) , and in this case , the maternal bonding comes through the mother s transmission line ( 0.38 ) and is characterized by high care ( 0.54 ) and low overprotection ( 0.71 ) .
similarly , as far as paternal bonding is concerned , class 1 is represented by the families of patients with anorexia ( 0.61 ) . in particular
, the paternal bonding comes through the father s transmission line ( 0.44 ) and is characterized by low care ( 0.81 ) and low overprotection ( 0.59 ) .
class 2 instead is represented by the control group ( 0.71 ) , where the paternal bonding comes through her mother s transmission line and is characterized by high care ( 0.79 ) and low overprotection ( 0.58 ) .
considering now the two groups , families of patients with anorexia versus control families , it is possible to evidence differences and analogies between them . for families of adolescents with anorexia ( class 1 ) ,
both maternal ( 0.52 ) and paternal ( 0.61 ) bonding comes through the father s transmission line ( 0.39 and 0.44 , respectively , for maternal and paternal bonding ) .
in fact , the adolescents father bonding is characterized by specificities concerning the relationship recalled both with his father and his mother .
however , the kind of transmission is not the same ; that is , maternal bonding is characterized by low care ( 0.75 ) and high overprotection ( 0.64 ) , whereas paternal bonding is characterized by low care ( 0.81 ) and low overprotection ( 0.59 ) . for control families ( class 2 ) ,
both maternal ( 0.59 ) and paternal ( 0.71 ) bonding comes through the mother s transmission line ( 0.38 and 0.46 , respectively , for maternal and paternal bonding ) .
the parental bonding recalled by the control adolescents mother is characterized by specificities both in the maternal and in the paternal bonding .
maternal and paternal bonding are characterized in fact by high care ( 0.54 and 0.79 , respectively ) and low overprotection ( 0.71 and 0.58 , respectively ) .
in the comparison between the families of patients with anorexia and the families belonging to the general population , the lca approach allowed to identify specific pattern of parental bonding transmission , separately for the maternal and the paternal bonding .
the first latent class was represented by the group of families of patients with anorexia and characterized by a low level of maternal care along with a high level of overprotection , which corresponds to the affectionless control parenting style.11 as far as adolescents with anorexia are directly concerned , it is worth noticing that they did not contribute significantly in defining this first class .
surprisingly , it was essentially the father to recall and perceive the mother as careless and overcontrolling
. however , the second profile ( representing the second latent class ) , specified by a high level of maternal care and a low level of protection ( corresponding to the optimal bonding),11 was characteristic of the control group ( table 2 ) and in particular of the control mothers .
the paternal bonding latent variable also presented two latent classes , which correspond respectively to the paternal bonding patterns of families of adolescents with anorexia and control families . a low level of paternal care characterized the first latent class , which specifies the families of patients with anorexia .
also in the case of paternal bonding , this profile did not receive a significant contribution from the adolescents with anorexia .
once again , however , the fathers of adolescents with anorexia perceived their own fathers as scarcely affectionate ; that is , they perceived an absent or weak bonding.11 conversely , the control families , and in particular the mothers , recalled their fathers as affectionate and warm toward them ; that is , they recalled an optimal bonding style.11 different from what has been shown for the maternal overprotection , the levels of paternal overprotection were not higher in families with adolescents with anorexia ( table 2 ) .
the similarity of results between the patients with restricting - type anorexia and the control adolescents ( tables 1 and 2 ) in the perceived maternal as well as paternal bonding style confirms the few previous findings in adolescents13,14 and actually raises the question of a potentially altered report of parental bonding due to the lack of awareness or specific personality traits of this kind of patients .
patients with restrictive conducts are in fact well known for their implacable attempt to deny difficulties , which can lead them even to death.13 it could be just because of their major concerns regarding autonomy and separation that adolescents with anorexia did not judge their parents less than perfect in the pbi questionnaire:13,14 perhaps , they may feel that a critique could be too much aggressive and concretely threatening the parents . in line with the defensive attitudes typical of the dismissing attachment , which has been shown to characterize many patients with restricting - type anorexia,8 idealization and avoidance
may thus prevent adolescents with anorexia from fighting with their parents , while many other adolescents , longing to win their autonomy , tend on the contrary to judge negatively parents and thus to overestimate the parental overprotection.13,25 the struggle for autonomy is indeed a key aspect characterizing the adolescent process of subjectivation and separation from the idealized parents of childhood , and at the same time , according to some theories , it is supposed to have a crucial role in eds psychopathology , where this physiological adolescent process may encounter some complications.26,27 on the other hand , it is also possible to explain the absence of differences between adolescents with anorexia and controls , assuming that patients with restricting - type anorexia show a parental bonding genuinely analogous to that of adolescents belonging to the general population .
this explanation seems however unlikely , based on the different findings obtained on patients with binge - eating / purging - type anorexia or with bulimia .
adolescent patients with binge - eating / purging - type anorexia in fact have been found to perceive their parental bonding as very inadequate with the mother as well as the father , described as excessively controlling and limiting the daughter s autonomy and freedom.14 it is worth noticing that these findings are reported from the same study that found , in line with our results , adolescent with restricting - type anorexia to describe parents as very caring and permissive , feeling an optimal bonding with them.14 the authors hypothesized that the difference between the two groups of patients had to be explained by the opposed defense and attachment mechanisms adopted by the purging ( more preoccupied ) and restricting ( more avoidant ) patients .
this conclusion may also be supported by the differences found in our sample between parents belonging to families of adolescents with anorexia and the parents belonging to the control group , where the bonding was perceived as more dysfunctional in the first group of parents . regarding the parents themselves ,
there has been little research specifically into the parents own perceived parental bonding , with the only exception of a study by canetti et al17 conducted exclusively on adult patients , which found some specificities on the paternal and above all on the maternal side . whether there might be intergenerational transmission of the attachment patterns along generations in patients with anorexia remains therefore still unclear .
overall , the results of the present study showed that the parents of adolescent patients with anorexia recall their own mothers as apprehensive and authoritarian ( high overprotection ) but not particularly affectionate and empathetic ( low care ) and their father as not particularly involved and affective in the children care ( low care ) .
in particular , the fathers relationships with their own parents were perceived as largely difficult , thus opening the hypothesis of a potential intergenerational transmission of the parental bonding in families of patients with anorexia , especially along the paternal line .
the inter - generational transmission of a less affective and highly protection - oriented quality of parental bonding may provide a vulnerable environment for the complex ethiopathogenesis of eds .
these findings open , but do not close , the question regarding the relationship between parenting styles in different generations and the development and course of anorexia nervosa in the offspring.17 according to the still very scarce and limited existing research on attachment states of mind in parents of patients with eds , mothers of patients with eds in fact showed an increased degree of unresolved state of mind concerning , in particular , losses and traumas , which have not yet been completely elaborated.17,28 an issue of unresolved loss appears to be consistent with the early separation difficulties that may be considered among the risk factors for the eds onset.28 according to the attachment theory , the representations and the internal working models of relationships can be transmitted through generations : the parents internal representation of relations with their own attachment figures may impact on their attitude toward their offspring , providing a sort of continuity across different generations parental behaviors . in line with these premises , in the only study specifically concerning the mothers of adult patients with anorexia , ward et al7 found a high rate of insecure attachment representation and unresolved losses , which are often connected to deficits and difficulties in the emotional regulation and reflective functioning.2,4 along with the attachment - related parenting behaviors , these latter characteristics may also in turn act as a risk factor in the mother
daughter relationship , potentially fostering the onset of an eating pathology in the daughters.3,7,28 maternal negative perception of their own mothers ( ie , pbi low levels of care ) seemed in fact to be linked with the severity of eating symptoms in the daughters with anorexia.17 consistent with these first findings on adults , the results of this study , conducted specifically on adolescents , may suggest that the intergenerational transmission of a less affective and highly protection - oriented quality of maternal bonding may provide a vulnerability to eds , whose multifactorial symptomatic manifestation is clearly precipitated further by different conditions and later circumstances.29 however , since it is impossible to draw causal inferences in this type of cross - sectional study , alternatively it is also plausible that the patients parents change their perception of their parents after the onset of the child s illness .
the deep sense of crisis and guilt linked to the child s condition may in fact lead parents to blame themselves and to perceive their own parental figures as inadequate as they feel to be at the moment for their ill offspring .
although previous studies have mainly explored the maternal side , our results showed an important contribution of the paternal perceptions in defining a specific profile of families with an adolescent offspring with anorexia , as if the male line would have a preeminent role in the perceptions of family dysfunctions .
a low level of care and affection perceived on the father s part in fact characterized the families of patients with anorexia .
this may imply that an intergenerational transmission of the parental bonding representations exists from father as well as from mother to offspring,17 suggesting that fathers perception of their own paternal figures involvement can play a pivotal role in their self - image as parents , and consequently influence the actual fathers behaviors with their offspring .
future studies will help to clarify this hypothesis , assessing not only the fathers perceptions of their own relationships with their parents but also the processes that link fathers evaluations of the parents attitudes with their own parenting representations and behaviors .
daughter relationship , recent studies have pointed to the equally important role of the father daughter bond as a risk or as protective factor in the onset and persistence of food - related symptoms.3032 if on the one hand the therapeutic alliance with parents has been demonstrated to predict a positive outcome of family therapies for adolescents with anorexia,32,33 on the other hand the father s exclusion may prevent treatment from success .
the results from this controlled study also support the major paternal contribution in the family dynamics of patients with anorexia,10,34,35 and therefore are likely to indicate that therapeutic approaches should include the paternal participation in the treatment and at the same time address issues related to the fathers perceptions of the parental role in the relationship with the adolescent with anorexia .
having said that , the limited number of patients and parents participating in the study , due to the particular clinical context of the research , imposes some limitations in interpreting the results of the lca applied .
moreover , the use of self - reported measures only , along with the cross - sectional design of the study , is a limitation that suggests caution in interpreting the results , without drawing any causal conclusions .
the self - reported retrospective measure of parental bonding has its undeniable advantages , such as the quickness to administer , and the information obtained directly from the individual concerned .
nevertheless , self - reported tools also have many weaknesses : the recall of the parental bonding is likely to be influenced by the subjective interpretation and modified by the real experiences of current relationships , particularly in the difficult and stressful situation of an anorexic onset.12 these measures are affected by the social desirability , which is particularly challenging in the case of patients with anorexia , which are well known for being inclined to provide idealized images , depleted from any negativity.13 moreover , it is worth saying that the results obtained concern specifically the particular western cultural context , in which the research has been conducted , since parenting styles differ greatly between cultures.3638 the pbi overprotection factor in fact showed some cross - cultural inconsistencies , since the more collective eastern cultures , and in particular the japanese and chinese ones , do not assign a positive value to child independence and autonomy.38 it is therefore likely that the positive link between high care and encouragement of freedom ( low overprotection ) may be specific only to western cultures , consequently representing a protective factor exclusively in these cultural contexts .
further research , conducted using a multi - informant assessment , based on the self - report tools along with clinician - report interviews , will help clarify the intergenerational transmission of the families attachment and bonding patterns and its relative role in the origin and course of anorexia nervosa .
this may help clinicians to provide more effective therapeutic treatments , tailored to the patient s and parents needs .
as opposed to making accusations for the offspring s difficulties,8,29 involving parents in the treatment of adolescent patients with anorexia and addressing how their own attachment39 and parental bonding affect current relationships could be helpful for the whole family dynamics and thus for the patient s outcome . | introductionthe attachment theory is widely used in order to explain anorexia nervosa origin , course and treatment response .
nevertheless , very little literature specifically investigated parental bonding in adolescents with anorexia , as well as the parents own bonding and intergenerational transmission within the family.purposethis study aims to identify any specific pattern of parental bonding in families of adolescents newly diagnosed with restricting - type anorexia , comparing them to the families of the control group.patients and methodsa total of 168 participants , adolescents and parents ( 78 belonging to the anorexia group and 90 to the control one ) , rated the perceived parental styles on the parental bonding instrument . the latent class analysis allowed the exploration of a maternal bonding latent variable and a paternal one.resultsthe main findings showed that a careless and overcontrolling parental style was recalled by the patients parents , and in particular by the fathers .
as far as the adolescents responses were concerned , patients with anorexia did not seem to express differently their parental bonding perception from participants of the control group.conclusionclinical implications driven from the results suggest that a therapeutic intervention working on how the parents own attachment representations influence current relationships may help to modify the actual family functioning and thus the outcome of patients with anorexia . |
epilepsy is caused by abnormal electrical discharges in a group of neurons in the brain and triggers repeated seizures ( 1 ) .
it is the second most important factor after stroke to cause the diseases of the central nervous system .
there are about 1.5 to 14 persons in every 1,000 individuals who are prone to epilepsy ( 3 ) .
epileptic patients , by comparison with normal individuals , experience more to health problems , low quality of life , emotional problems , physical or mental difficulties ( 4 , 5 ) , depression , anxiety ( 6 - 9 ) , problems relating to emotional and physical health ( 8 - 14 ) . according to the world health organization , mental health is a state of well - being and recovery in which an individuals can accomplish his potential , cope with stress , act in a fruitful manner , and play an effective role in the community ( 15 ) .
in addition to psychological problems , epileptic individuals have negative self - perception and tend to attribute their failures to their insufficiencies ; the failures which usually are the consequences of a neurological disease .
they spread their inefficient feelings to their other experiences and , as a result , rather than address the problem appropriately , experience isolation and helplessness .
according to the revised helplessness theory ( 16 ) , individuals attribute their helplessness to the situations which are categorized in 3 dimensions : internal - external , general - specific , and stable - unstable ( 17 ) .
seligman stated that not only lack of control in terms of learned helplessness influences human health and well - being , but also it is important that how a person explains and attributes the lack of control ( 18 ) .
there are 2 main attributional styles : pessimistic and optimistic . in the optimistic attributional style ,
negative or unpleasant events are attributed to external , unstable , and specific factors , while in the pessimistic attributional style , internal , unstable , and general factors are considered as the causal factors ( 19 ) .
cognitive and meta - cognitive methods have been used to modify the attributional style ( 20 ) . amongst those methods ,
attribution retraining is one of the most efficient methods in that it aims at enabling individuals to reconstruct their causal explanations of failures ( 21 ) .
different studies have suggested that the attributional style influences the physical and psychological consequences of negative life events ( 22 ) .
studies have shown that attribution retraining reduces physical problems ( 23 - 25 ) , depression ( 24 ) , and anxiety ( 24 ) and increases performance ( 26 , 27 ) .
as one of the main concerns in every country is to guarantee the mental health of children , especially epileptic ones , the application of such programs as attribution retraining could play an effective role in the prevention of problems relating to physical and psychological problems .
this study was conducted to assess the efficacy of attribution retraining on promoting mental health and its components ( physical symptoms , social dysfunction , anxiety and insomnia , and depression ) in epileptic children .
the present study is a semi - experimental investigation with a pretest and posttest design and includes a control group . from 175 epileptic children who were registered at the iranian epilepsy association ( iea ) in tehran in 2014 ,
24 children were excluded from the study due to physical and motor inabilities , sensory difficulties ( blindness and deafness ) , behavior disorders ( conduct disorder , disruptive behavior disorder , and oppositional conduct disorder ) , and participation in a similar program at the same time . from the remaining 151 children , 30 children ( 17 boys and 13 girls ;
mean standard deviation = 15.87 1.41 , respectively ) were selected randomly by lottery and according to our sample size formula ( = 0.05 , expected power = 0.80 , standard deviation = 15 , and mean deviation = 11 ) and inclusion criteria . the iea is a charity , and our subjects were registered there and received free medical and psychological services .
subjects were included according to their age ( 14 - 18 years old ) and controlled epilepsy ( having received medication for the preceding 6 months and without seizure ) .
all the subjects completed the general health questionnaire ( ghq ; goldberg and hiller , 1979 ) before and after the training sessions .
this is a screening , self - reporting questionnaire which was constructed by goldberg and hiller ( 1979 ) and has been used in clinical settings with the aim of differentiating people with mental disorders ( 28 ) .
the questionnaire has 4 subscales of physical symptoms , anxiety and insomnia , social dysfunction , and depression .
each subscale has 7 items , and the sum of the scores of the 4 subscales yields an index for general mental health .
taghavi reported its reliability through test - retest , two - split , and cronbach s alpha of 0.70 , 0.93 , and 0.90 , respectively .
receiving 6 points in each subscale and 22 points and higher for the total scale is an indicator for psychopathology ( 29 ) .
the study was formally approved by the ethics committee of university of social welfare and rehabilitation sciences in iran . written informed consent was obtained from the children s parents .
thereafter , 30 epileptic children were selected randomly from those at the iea in tehran , 2013 .
subjects were selected according to our sample size formula and inclusion and exclusion criteria and were assigned to experimental and control groups in equal numbers .
the experimental group participated in 11 training sessions ( twice a week ; 45 minutes per session ) and received an attribution retraining program in addition to a routine program , whereas the control group received only the routine program ( i.e. , communication training , anger management , and life skills training ) presented in the iea . after the eleventh session and subsequently 6 weeks later , the general health questionnaire was completed by all the subjects .
the data were analyzed via the analysis of covariance ( ancova ) using statistical package for the social sciences ( spss ) , version 16 .
the attribution retraining program was set based on the bandura theory about self - efficacy , the seligman learned helplessness model , and the wiener attribution model .
this package was set by golparvar ( 30 ) for students with dyscalculia for the first time in iran .
the reliability and validity of the package were reported to be 0.92 and 0.86 , correspondingly .
the contents of each sessions were as follows ( table 1 ) : the flow chart of the study is as follows : literature review ( 2 months ) investigating the population and sample selection ( 1 month ) administrating the intervention and data collection ( 3 months ) entering the data into spss and data analysis ( 1 month ) writing and editing the project and providing the paper ( 4 months )
from 175 epileptic children who were registered at the iranian epilepsy association ( iea ) in tehran in 2014 , 24 children were excluded from the study due to physical and motor inabilities , sensory difficulties ( blindness and deafness ) , behavior disorders ( conduct disorder , disruptive behavior disorder , and oppositional conduct disorder ) , and participation in a similar program at the same time . from the remaining 151 children , 30 children ( 17 boys and 13 girls ;
mean standard deviation = 15.87 1.41 , respectively ) were selected randomly by lottery and according to our sample size formula ( = 0.05 , expected power = 0.80 , standard deviation = 15 , and mean deviation = 11 ) and inclusion criteria . the iea is a charity , and our subjects were registered there and received free medical and psychological services .
subjects were included according to their age ( 14 - 18 years old ) and controlled epilepsy ( having received medication for the preceding 6 months and without seizure ) .
all the subjects completed the general health questionnaire ( ghq ; goldberg and hiller , 1979 ) before and after the training sessions .
this is a screening , self - reporting questionnaire which was constructed by goldberg and hiller ( 1979 ) and has been used in clinical settings with the aim of differentiating people with mental disorders ( 28 ) .
the questionnaire has 4 subscales of physical symptoms , anxiety and insomnia , social dysfunction , and depression .
each subscale has 7 items , and the sum of the scores of the 4 subscales yields an index for general mental health .
taghavi reported its reliability through test - retest , two - split , and cronbach s alpha of 0.70 , 0.93 , and 0.90 , respectively .
receiving 6 points in each subscale and 22 points and higher for the total scale is an indicator for psychopathology ( 29 ) .
the study was formally approved by the ethics committee of university of social welfare and rehabilitation sciences in iran . written informed consent was obtained from the children s parents .
thereafter , 30 epileptic children were selected randomly from those at the iea in tehran , 2013 .
subjects were selected according to our sample size formula and inclusion and exclusion criteria and were assigned to experimental and control groups in equal numbers .
the experimental group participated in 11 training sessions ( twice a week ; 45 minutes per session ) and received an attribution retraining program in addition to a routine program , whereas the control group received only the routine program ( i.e. , communication training , anger management , and life skills training ) presented in the iea .
after the eleventh session and subsequently 6 weeks later , the general health questionnaire was completed by all the subjects .
the data were analyzed via the analysis of covariance ( ancova ) using statistical package for the social sciences ( spss ) , version 16 .
the attribution retraining program was set based on the bandura theory about self - efficacy , the seligman learned helplessness model , and the wiener attribution model .
this package was set by golparvar ( 30 ) for students with dyscalculia for the first time in iran .
the reliability and validity of the package were reported to be 0.92 and 0.86 , correspondingly .
the contents of each sessions were as follows ( table 1 ) : the flow chart of the study is as follows : literature review ( 2 months ) investigating the population and sample selection ( 1 month ) administrating the intervention and data collection ( 3 months ) entering the data into spss and data analysis ( 1 month ) writing and editing the project and providing the paper ( 4 months )
the descriptive indices of the experimental and control groups according to their sex and age are depicted in table 2 .
the results of the test showed no significant difference between the experimental and control groups according to gender and age .
the mean standard deviation of mental health and its components for the experimental and control groups in the pretest and posttest separately is presented in table 3 . as is shown in table 3
, there was a difference between the mean scores of the experimental and control groups in terms of mental health and its components .
it was required to test the assumption of the ancova to determine the effectiveness of attribution retraining on mental health in the epileptic children .
smirnov test ( table 4 ) demonstrated that all the variables were normal , and the leven test was not significant ( p > 0.01 ) ( table 5 ) .
abbreviation : f , f statistics which is calculated according to the ratio of two variances .
also , the diagram of regression square linear was shown to be 0.106 , denoting a linear relation between the pretest and posttest of mental health in the 2 groups .
consequently , is was possible to use the ancova since all its assumptions were tested .
the ancova was utilized to compare mental health between the experimental and control groups in posttest ( table 6 ) . as is shown in table 6 , mental health and its components ( physical symptoms , anxiety and insomnia , social dysfunction , and depression )
were different between the experimental and control groups significantly ( p < 0.01 ) . according to the eta quotient
, it can be concluded that 43% , 62% , 55% , 78% , and 65% of the variances of physical symptoms , anxiety and insomnia , social dysfunction , depression , and mental health were explained by attending the attribution retraining sessions .
also , there were no significant differences between the scores of mental health and its components in the posttest and follow - up .
the major finding of the present study was that physical symptoms in the epileptic children decreased significantly after participating in the attribution retraining sessions .
this finding was concordant with the results of some previous studies ( 31 , 32 ) .
they tend to attribute their failures ( which are partly because of having a neurological disease ) to their incompetence , and then spread their feelings of inadequacy and helplessness to their other experiences .
consequently , they miss the opportunities to perform effectively and finally yield to loneliness and helplessness rather than seek to address the problem appropriately . in the present study , it seems that modification from the pessimistic attributional style to the optimistic attributional style , which occurred in the intervention sessions , caused the epileptic children to attribute unpleasant events to unstable , specific , and external factors . as a result , they experienced fewer physical symptoms than before ( 19 ) .
another finding in the current study suggested that the attribution retraining program reduced anxiety and insomnia in the epileptic children , which chimes in with the results of a study by larisch et al .
( 24 ) , who concluded that attribution retraining led to a reduction in anxiety and insomnia in individuals with health problems .
epilepsy can affect adaptive skills and coping with problems pertaining to a chronic disease through creating inefficient underlying beliefs .
as these beliefs are different from those of normal individuals and are reinforced by negative experiences allied to epilepsy , they will generate high levels of chronic anxiety ( 33 , 34 ) .
attribution retraining programs may result in more social relations , better mood , and modification of children s attitude toward themselves and their abilities . amongst our study population , the attributional style of the epileptic children
may have been altered from external , general , and stable dimensions to external , specific , and unstable ones ( 35 ) and , thus , brought about an amelioration in their anxiety .
this finding is in line with the results of some previous studies ( 26 , 27 , 31 , 35 ) .
negative attitudes toward epilepsy can cause social maladjustment ; these children , therefore , experience problems in interpersonal relations because of the nature of their illness . since correcting the communication style ( often aggressive or passive ) was the purpose of the program in the current study
, the subjects may have been motivated to improve their social functioning ( 36 ) .
individuals harboring negative expectations and beliefs toward themselves and their current position are liable to interpret events pessimistically , have low performance , predict the future negatively , and attribute their performance to negative causes .
furthermore , low expectations of success lessen individuals motivation for participating in social activities ( 37 ) . in the present study
, it seems that the attribution retraining program enhanced social performance in the children through modifying their pessimistic attributional style to the optimistic attributional style .
layden ( 37 ) , larisch ( 24 ) and yahyaee ( 31 ) stated that epileptic children are repeatedly exposed to seizures ( as an unpredictable and uncontrollable event ) , so they gradually experience helplessness and passivity .
they often fear being invaded by social environment and are nervous of other people s misinterpretation of them , which precludes them from having positive experiences . in sum , isolation and passivity can affect individuals self - perception and lead to feeling of inadequacy , helplessness , and depression . considering that attribution retraining is a method for modifying individuals perception about the causes of success and failure
an appropriate training method should address the concept of attributional style , causes of events in different situations , effects of attributions on prospective expectations , and disruptive effects of unstable and internal attributions vis - - vis failure . in the current study
, attribution retraining may have been able to play a major role in familiarizing the epileptic children with the positive effects of attributional styles on health and in developing their skills for confronting events and , thus , reduce their depression .
therefore , this method may enable individuals to correct the negative thoughts and inefficient beliefs that underlie their depression ( 36 ) .
this finding is in concordance with the results of some previous studies ( 31 , 32 , 34 , 36 , 38 , 39 ) .
iravani and izadi found a relationship between attributional styles and mental health in college students .
the present finding can be explained in 3 ways : 1 ) the pessimistic attributional style renders individuals prone to helplessness , which results in cognitive deficits as well as emotional , social , and physical disabilities and ultimately health problems .
2 ) pessimists are not aware of the consequences of their behaviors and fail to make the necessary effort to improve their situation , which will worsen their mental health .
3 ) it is plausible that the pessimistic attributional style may be due to illness and enormous mental health problems .
the notion that pessimists , in comparison to optimists , are susceptible to mental health problems can be explained in 3 ways .
firstly , the pessimistic attributional style leads to helplessness , the effects of which manifest as cognitive , affective , social , and physical disorders or deficiencies and these deficiencies will ultimately cause health problems .
secondly , pessimists are oblivious to the consequences of their behaviors and do not seek to adequately alter their behaviors ; consequently , they are liable to develop health problems . and thirdly , it is widely acceptable that the pessimistic attributional style may be the outcome of disease or health problems ( 40 ) . in the current study
, the results of follow - up showed no significant difference in attributional styles between the experimental and control groups . to explain this finding
, one should take into account the fact that attribution retraining requires that children cooperate actively to recognize maladaptive and negative attributions and show sufficient motivation to modify these attributions ; it is probable that the children in our study failed to accomplish their assignments in the time interval between the termination of the program and follow - up evaluation .
moreover , the preservation of mental health needs more time than the few intervention sessions made available to the epileptic children in the present study . according to the wiener theory ( 40 ) ,
it is suitable to attribute successes to internal , stable , and general factors and failures to external , unstable , and specific causes . in the present study ,
also , our findings showed that the attribution retraining program was effective in restructuring the epileptic children causal explanation of failures . indeed , the children succeeded in attributing their successes to internal , stable , and general factors and their failures to external , unstable , and specific factors .
so , the program was able to improve mental health in the epileptic children in our study .
the attribution retraining program can be employed by educators , psychologists , and counselors who provide rehabilitation services for epileptic children .
it can enhance quality of life and mental health in children and prevent probable adaptive problems in adulthood .
our study had some limitations , first and foremost amongst which was its low sample size .
we reported the effectiveness of attribution retraining on mental health in epileptic children aged between 14 and 18 years who lived in tehran .
consequently , a population - based study is required to determine the effectiveness of attribution retraining on mental health in all iranian children with epilepsy for an appropriate interpretation of the results .
in fact , there is a need to assess the status of mental health in the pediatric population via the same method .
the distribution of the intervention sessions in 11 weeks ( 1 session per week ) may augment the positive effect of intervention on mental health at follow - up . as the sessions were held twice a week in our study
, it is probable that the children were dependent on the trainer s supervision and did not feel responsible for accomplishing their tasks . according to a previous study ,
a 20-week program had a positive impact on mental health in the study population in the long term , even at 6 weeks follow - up ( 41 ) .
the strong point of the present study was the application of attribution retraining on epileptic children before 18 years old .
it is deserving of note that the implementation of a similar intervention for rehabilitation purposes for epileptic children could confer better mental health by preventing many health problems that may interfere with epilepsy .
we recommend that future studies recruit larger sample volumes , comprising subjects of both sexes and different age brackets , so as to better evaluate the effectiveness of attribution retraining on boys and girls at different ages .
attribution retraining can be applied as an effective intervention by experts , teachers , and parents .
the program can be implemented in family training classes , so that parents will be able to supervise the accomplishment of the assignments by their children . | background : epilepsy affects children s quality of life and leads to social and mental problems . promoting the mental health of children , especially epileptic ones , and preventing problems affecting them constitute major concerns for every country .
mental health promotion requires intervention programs.objectives:we sought to assess the efficacy of attribution retraining on the mental health of epileptic children.patients and methods : the present study is a semi - experimental investigation with a pretest and posttest design and includes a control group .
thirty children , comprising 17 boys and 13 girls , were selected randomly from the iranian epilepsy association in tehran and assigned to experimental and control groups .
they answered to the general health questionnaire ( goldberg and hiller , 1979 ) .
the experimental group participated in 11 training sessions ( twice a week ; 45 minutes for each session ) and received attribution retraining .
the data were analyzed using the multiple analysis of covariance.results:the findings showed that the experimental group , in comparison with the control group , experienced a reduction in physical symptoms , anxiety and insomnia , social dysfunction , and depression and an increase in mental health significantly ( p < 0.01 ) after the training sessions .
there were no significant differences , however , between the two groups at 6 weeks follow-up.conclusions:attribution retraining improved mental health in the epileptic children in our study .
it , therefore , seems to be an appropriate intervention for promoting the mental health of children . |
in a phase ii multicenter double - blind study of gadobenate dimeglumine ( gd - bopta ; bracco spa , milan , italy ) , 113 adult patients with known or suspected liver masses were examined . from this group , 31 patients with biopsy proven malignant primary liver tumors ( hepatocellular carcinoma in 29 and cholangiocarcinoma in 2 ) were identified .
there were 8 women and 23 men with a mean age of 59 years ( range 32 to 75 years ) . in 14 patients there was biopsy or imaging evidence of cirrhosis .
based on a reference standard imaging , a total of 71 focal lesions were identified in these patients . the presence and absence of a liver lesion
was confirmed by intra operative ultrasound ( ious ) in 24 patients ( 48 lesions ) , computed tomography during arterial portography ( ctap ) in 4 patients ( 15 lesions ) , and lipiodol ct ( ctlp ) in 3 patients ( 8 lesions ) .
all patients received at least one of these reference standard imaging studies of the liver within 7 days before to 14 days after the mr imaging . in patients with more than one reference standard
a cyst and two focal areas of focal nodular hyperplasia were identified and excluded from further analysis .
gadobenate dimeglumine is an octadentate chelate of gadolinium which may be used as a non - specific contrast agent like the conventional gadolinium agents as well as a liver - specific contrast agent ( 11 ) .
the chemical and pharmacodynamic properties of this contrast agent have been described previously ( 12 - 16 ) . in brief , following rapid ( 2 ml / min ) intravenous injection of the contrast agent , it initially redistributes into the extracellular space and is then largely eliminated by the kidneys .
some 3 - 5% of the injected dose is however taken up by functioning hepatocytes and undergoes biliary excretion . despite the low percentage of hepatobiliary excretion ,
the fraction taken up produces clinically useful hepatic enhancement that increases liver signal - to - noise ratio ( snr ) and liver - to - lesion contrast - to - noise ratio ( cnr ) ( 6 - 8 , 14 ) . peak enhancement of lesion - to - liver contrast occurs between 40 and 120 minutes . since one goal of the clinical trial was to determine optimal dose ,
the contrast agent ( either 0.25 mmol / l or 0.5 mmol / l solution ) was administered intravenously at 10 ml / min immediately after the unenhanced mr examination .
seventeen patients received 0.05 mmol / kg , and 14 patients received 0.10 mmol / kg .
post contrast imaging was performed at two delayed time points in order to determine the optimal imaging period in the hepatocyte phase .
mr imaging was performed at 1.5 tesla with t1-weighted spin echo ( se ) and t1-weighted gradient echo ( gre ) pulse sequences before contrast administration as well as 40 - 80 minutes ( early ) and 90 - 120 minutes ( late ) after contrast administration .
the liver was imaged with 10 - 14 axial 10 mm thick sections with 2 mm gaps ; a matrix size of 140 - 160256 , and a tailored field of view ranging from 285 - 350380 - 400 mm . t1-weighted se sequences utilized a repetition time between 350 - 550 msec and an echo time of 15 msec and 3 - 4 excitations .
the t1-weighted gre images were acquired with a repetition time of 95 - 150 msec , a 4 msec echo time , an 800 flip angle and one excitation during a single breath hold .
three off - site independent reviewers ( a , b , c ) evaluated the mr images separately in a random unpaired fashion that included the pre contrast images and the post contrast images .
each set of mr images was evaluated individually and the number , size , and location by surgical segment of lesions were recorded on liver maps . additionally , each reviewer , using a 5-point scale system , documented the confidence with which a lesion was detected .
a point of 1 indicated that the lesion was definitely present , a point of 2 indicated that a lesion was probably present while a point of 3 indicated an indeterminate lesion .
point of 4 and 5 indicated that a lesion was probably not present or definitely not present respectively .
each reviewer assessed the presence of lesions for each patient at each time point for each sequence .
the total number of lesions as well as their size and location was determined from the reference standard imaging . in a patient with multiple lesions ,
no more than five lesions per patient were assessed in order not to bias the findings toward patients with multiple lesions .
this allowed inclusion of all patients without skewing the data from the patients with many lesions .
a lesion was considered present if the reviewer confidence ranking was either a 1 or 2 point ( definitely or probably present ) .
false positive lesions were defined as lesions reported by the reviewers with a confidence of 1 or 2 with no corresponding lesion by the reference standard .
analysis according to lesion size was performed by dividing the lesions into three groups based on diameter size : 1 cm or less ; between 1 cm to 2 cm ; and greater than 2 cm . the efficacy of gadobenate dimeglumine enhanced mri was evaluated by assessing the change in detection ( relative to the reference standard ) from pre - contrast mri to contrast enhanced mri .
the change in concordance with the reference standard from unenhanced to enhanced mri in the determination of the number of focal liver lesions was tested using a two tailed mcnemars test .
differences in concordance were also studied between concentrations and imaging times using a two tailed mcnemars test . because all patients were selected on the basis of lesion pathology , specificity and accuracy
in a phase ii multicenter double - blind study of gadobenate dimeglumine ( gd - bopta ; bracco spa , milan , italy ) , 113 adult patients with known or suspected liver masses were examined . from this group , 31 patients with biopsy proven malignant primary liver tumors ( hepatocellular carcinoma in 29 and cholangiocarcinoma in 2 ) were identified .
there were 8 women and 23 men with a mean age of 59 years ( range 32 to 75 years ) . in 14 patients there was biopsy or imaging evidence of cirrhosis .
based on a reference standard imaging , a total of 71 focal lesions were identified in these patients . the presence and absence of a liver lesion
was confirmed by intra operative ultrasound ( ious ) in 24 patients ( 48 lesions ) , computed tomography during arterial portography ( ctap ) in 4 patients ( 15 lesions ) , and lipiodol ct ( ctlp ) in 3 patients ( 8 lesions ) .
all patients received at least one of these reference standard imaging studies of the liver within 7 days before to 14 days after the mr imaging . in patients with more than one reference standard
a cyst and two focal areas of focal nodular hyperplasia were identified and excluded from further analysis .
gadobenate dimeglumine is an octadentate chelate of gadolinium which may be used as a non - specific contrast agent like the conventional gadolinium agents as well as a liver - specific contrast agent ( 11 ) .
the chemical and pharmacodynamic properties of this contrast agent have been described previously ( 12 - 16 ) . in brief , following rapid ( 2 ml / min ) intravenous injection of the contrast agent , it initially redistributes into the extracellular space and is then largely eliminated by the kidneys .
some 3 - 5% of the injected dose is however taken up by functioning hepatocytes and undergoes biliary excretion . despite the low percentage of hepatobiliary excretion ,
the fraction taken up produces clinically useful hepatic enhancement that increases liver signal - to - noise ratio ( snr ) and liver - to - lesion contrast - to - noise ratio ( cnr ) ( 6 - 8 , 14 ) .
peak enhancement of lesion - to - liver contrast occurs between 40 and 120 minutes . since one goal of the clinical trial was to determine optimal dose ,
the contrast agent ( either 0.25 mmol / l or 0.5 mmol / l solution ) was administered intravenously at 10 ml / min immediately after the unenhanced mr examination .
seventeen patients received 0.05 mmol / kg , and 14 patients received 0.10 mmol / kg .
post contrast imaging was performed at two delayed time points in order to determine the optimal imaging period in the hepatocyte phase .
mr imaging was performed at 1.5 tesla with t1-weighted spin echo ( se ) and t1-weighted gradient echo ( gre ) pulse sequences before contrast administration as well as 40 - 80 minutes ( early ) and 90 - 120 minutes ( late ) after contrast administration .
the liver was imaged with 10 - 14 axial 10 mm thick sections with 2 mm gaps ; a matrix size of 140 - 160256 , and a tailored field of view ranging from 285 - 350380 - 400 mm .
t1-weighted se sequences utilized a repetition time between 350 - 550 msec and an echo time of 15 msec and 3 - 4 excitations .
the t1-weighted gre images were acquired with a repetition time of 95 - 150 msec , a 4 msec echo time , an 800 flip angle and one excitation during a single breath hold .
three off - site independent reviewers ( a , b , c ) evaluated the mr images separately in a random unpaired fashion that included the pre contrast images and the post contrast images .
each set of mr images was evaluated individually and the number , size , and location by surgical segment of lesions were recorded on liver maps . additionally , each reviewer , using a 5-point scale system , documented the confidence with which a lesion was detected .
a point of 1 indicated that the lesion was definitely present , a point of 2 indicated that a lesion was probably present while a point of 3 indicated an indeterminate lesion .
point of 4 and 5 indicated that a lesion was probably not present or definitely not present respectively .
each reviewer assessed the presence of lesions for each patient at each time point for each sequence .
the total number of lesions as well as their size and location was determined from the reference standard imaging . in a patient with multiple lesions ,
no more than five lesions per patient were assessed in order not to bias the findings toward patients with multiple lesions .
this allowed inclusion of all patients without skewing the data from the patients with many lesions .
a lesion was considered present if the reviewer confidence ranking was either a 1 or 2 point ( definitely or probably present ) .
false positive lesions were defined as lesions reported by the reviewers with a confidence of 1 or 2 with no corresponding lesion by the reference standard .
analysis according to lesion size was performed by dividing the lesions into three groups based on diameter size : 1 cm or less ; between 1 cm to 2 cm ; and greater than 2 cm .
the efficacy of gadobenate dimeglumine enhanced mri was evaluated by assessing the change in detection ( relative to the reference standard ) from pre - contrast mri to contrast enhanced mri .
the change in concordance with the reference standard from unenhanced to enhanced mri in the determination of the number of focal liver lesions was tested using a two tailed mcnemars test .
differences in concordance were also studied between concentrations and imaging times using a two tailed mcnemars test . because all patients were selected on the basis of lesion pathology , specificity and accuracy could not be assessed .
the number of focal liver lesions identified on gd - bopta enhanced mr images exceeded the number identified on unenhanced images ( table 1 ) .
table 1 separates the number of lesions identified by reviewer by time point as well as by imaging sequence . by combining both the t1-weighted gre and t1-weighted se images before and after contrast ( both early and late time points ) for reviewers a ,
b and c , the number of lesions identified increased after contrast administration from 34 to 53 , 37 to 48 , and 43 to 49 , respectively .
there was no significant difference in lesion detection between the two concentrations ( 0.05 mmol / kg and 0.10 mmol / kg ) of contrast administered .
table 1 shows that , in general , more lesions were identified with the t1-weighted gre pulse sequence when compared to the t1-weighted se technique , and more lesions were identified after contrast administration when both imaging techniques were analyzed together .
most lesions were identified on the late imaging time point ( 90 - 120 minutes ) although several more lesions ( total number ) were identified combining both the early and late post - contrast images .
the number of false positive lesions decreased for two of the three reviewers after the administration of gd - bopta ( table 2 ) .
one reviewer detected all of these lesions while the other two reviewers detected 14 out of 15 solitary lesions .
there were 5 patients with 2 lesions ; both lesions were identified only after contrast administration in 4 of the 5 patients . in the remaining patient with two lesions , the second lesion ( 25 mm ) was not identified before or after contrast . in two of these patients gadobenate dimeglumine administration allowed identification of the second lesion while in the other two patients , gd - bopta administration was required for the visualization of both ( fig .
one lesion was identified before and after gadobenate dimeglumine in all of these patients ; however , the second and third lesions were only identified by one of the reviewers in one patient and two reviewers in another patient . in the third patient with three lesions ,
two out of three reviewers detected more lesions after contrast administration in 6 out of these 8 patients ( fig 2 ) . in the remaining two patients , one patient had no additional lesions identified while only 1 out of the 3 reviewers detected more lesions after contrast in the other patient . in 68% of the patients with more than one lesion by reference standard ( 11 of 16 patients ) ,
after contrast administration , smaller lesions were better detected by two of the three reviewers ( table 3 ) . from a total of 29 lesions smaller than 1 cm ,
the number of lesions detected before and after contrast infusion for reviewers a , b , and c where 7 to 21 , 9 to 15 , and 16 to 18 respectively .
this was statistically significant ( p<0.05 ) for only two of the three reviewers ( fig 3 ) . the number of lesions detected for both the 1 - 2 cm and greater than 2 cm group also increased after contrast administration .
this study shows that gadobenate dimeglumine enhanced t1-weighted mr imaging can improve the detection of primary hepatic malignant neoplasms .
our findings confirm prior reports that have noted improvement in lesion - liver contrast on gd - bopta enhanced images .
furthermore , our findings show that the most important benefit is in the detection of small ( less than 1 cm ) tumors .
prior reports have provided conflicting findings on contrast enhanced mr imaging using hepatocyte targeted contrast agents in patients with cirrhosis .
for example , murakami et al . noted that compared to normal liver , patients with cirrhosis had lower hepatic enhancement with mn - dpdp ( 9 ) while manfredi et al
. reported similar enhancement between normal and cirrhotic livers with gadobenate dimeglumine ( 6 ) .
these differences may be due to variations in the severity of cirrhosis in the subject groups or due to differences in pharmacokinetics of the two drugs as gd - bopta also shows non - specific tissue enhancement ( extracellular effect ) in addition to hepatocyte specific liver enhancement .
our results have important implications with respect to imaging technique and the evaluation of patients with primary hepatic neoplasms .
firstly t1-weighted gre techniques were superior compared to se techniques and there was only minimal improvement in the total number of lesions detected when both sequences were used together .
prior studies have in fact reported that quantitative measurement of liver - to - lesion contrast on t1-weighted gre images is superior to that on se images ( 17 ) .
this advantage is most likely related to greater t1-weighting on the gre images due to the availability of shorter tes and to decreased motion - related noise in these breath - hold images .
these images can also be obtained at thinner section ( 8 mm ) with ample coverage to study the entire liver . while use of fatsaturation may also provide additional benefit , this was not studied and requires future attention ( 18 ) . in terms of the optimal imaging window
, it appears there is a favorable distinction between liver and primary malignant hepatic neoplasms at both the early and late time points after gadobenate dimeglumine administration although slightly more total lesions were detected at the late time point ( 90 - 120 minutes after injection ) .
there was no significant difference in lesion detection between the two doses ( 0.05 mmol / kg and 0.10 mmol / kg ) evaluated .
one possible way to image with this agent is to initially perform dynamic imaging immediately following the pre - contrast examination ( not performed in these cases ) and then , if needed , have the patient return at a convenient time between 1 - 2 hours later to have delayed scans performed .
this approach may not only increase the yield in lesion detection in patients with cirrhosis where dynamic scanning is known to be of critical importance , but may also provide important perfusion data for tissue characterization .
moreover , this approach may well be the preferred procedure in patients suspected of harboring liver metastases .
in such patients , dynamic imaging with conventional gadolinium agents does not necessarily improve lesion detectability over unenhanced scans alone ( 19 - 20 ) .
this did not allow determination of accuracy , sensitivity or specificity since there was an unintentional bias as the reviewers expected to identify at least one hepatic mass .
in addition the off - site analysis methodology may also have biased the results against gd - bopta since clinical information may have improved diagnostic performance was not available . furthermore , the presence of cirrhosis was not proven in all subjects ; it is likely that this patient population represented a spectrum of underlying chronic liver disease and extrapolation to a cirrhotic population should be made with caution .
another limitation was that even though histological diagnosis was available for all the patients , not every lesion was sampled .
finally , comparison of this agent to contrast - enhanced helical ct may also be necessary to determine the clinical utility of gadobenate dimeglumine enhanced liver mr imaging .
clinical utility may also be affected by the fact that the t2-weighted images were not utilized for the evaluation of the images .
this methodology was adopted in order to best evaluate the effect of the contrast agent on t1-weighted images . however , in actual practice , patients would not be evaluated without simultaneous review of the t2-weighted images . in conclusion ,
gd - bopta may serve a role as a contrast agent to identify primary malignant hepatic neoplasms with mr imaging . from our
blinded off - site multicenter evaluation , the contrast agent aids in the detection of smaller lesions in a patient group where traditional imaging ( us , ct , mr ) has been limited . | objectiveto investigate the efficacy of gadobenate dimeglumine ( gd - bopta ) enhanced mr imaging for the detection of liver lesions in patients with primary malignant hepatic neoplasms.materials and methodsthirty - one patients with histologically proven primary malignancy of the liver were evaluated before and after administration of gd - bopta at dose 0.05 or 0.10 mmol / kg .
t1-weighted spin echo ( t1w - se ) and gradient echo ( t1w - gre ) images were evaluated for lesion number , location , size and confidence by three off - site independent reviewers and the findings were compared to reference standard imaging ( intraoperative ultrasound , computed tomography during arterial portography or lipiodol computed tomography ) .
results were analyzed for significance using a two - sided mcnemar 's test.resultsmore lesions were identified on gd - bopta enhanced images than on unenhanced images and there was no significant difference in lesion detection between either concentration .
the largest benefit was in detection of lesions under 1 cm in size ( 7 to 21 , 9 to 15 , 16 to 18 for reviewers a , b , c respectively ) . in 68% of the patients with more than one lesion ,
gd - bopta increased the number of lesions detected.conclusionliver mr imaging after gd - bopta increases the detection of liver lesions in patients with primary malignant hepatic neoplasm . |
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