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hepatocellular carcinoma ( hcc ) is one of the most common cancers worldwide , where it represents the third cause of cancer - related death . because of differences in risk factors ,
it ranges from 5.5 to 14.9 per 100,000 individuals , but it may reach 100 per 100,000 in some regions of africa and south east asia .
hepatitis b virus ( hbv ) infection is the main risk factor in asia and africa , whereas hepatitis c virus ( hcv ) infection is the main risk factor in western countries and in japan .
other conditions increasing the risk of chronic liver disease and hcc development are alcohol abuse , aflatoxin b1 or vinyl chloride exposure , primary biliary cirrhosis , diabetes , non - alcoholic fatty liver disease and genetic disorders like haemochromatosis and 1-antitrypsin deficiency .
many of these factors are known causes of liver cirrhosis , which represents a pre - neoplastic condition for hcc .
the remaining hccs , which arise in an otherwise healthy liver , are thought to develop , at least in part , from the malignant transformation of liver adenomas .
a worldwide accepted staging system for hcc on cirrhosis , able to provide the proper guide for treatment and overall management of cirrhotic and hcc patients , is lacking . among the several proposed hcc classification systems ,
the barcelona clinic liver cancer ( bclc ) ( table 1 ) is one of the most used , as it offers the advantage of linking tumour staging to the best therapeutic strategy .
hepatocellular carcinoma classification according to the bclc staging system treatment strategy is tailored on the basis of the extent of tumour burden , liver function , physical status and potential treatment efficacy . in very early and early stage hcc , potential curative treatments are available .
they include surgical resection , percutaneous ablation and liver transplantation . however , only about 3040% of cirrhotic patients enrolled in surveillance programs are eligible for these types of intervention and , even after a curative treatment , the recurrence rate approaches 70% at 5 years . in advanced hcc ,
treatment options are even more limited : curative treatments are not available and traditional chemotherapy proved to be only marginally effective or even toxic in either adjuvant or neo - adjuvant settings .
thus , the identification of new possible targets for the development of non - conventional treatments is urgent and will necessarily take advantage of progresses in the comprehension of the molecular pathogenesis of hcc .
initial steps involve the disruption of a set of interdependent pathways controlling the homeosta - sis between cell growth and apoptosis . at later stages
, cells may acquire angiogenic , invasive and metastatic properties , in a process that involves the interactions of neoplastic cells with the surrounding microenvironment . to develop the above cancer traits , several elements of the retinoblastoma ( rb ) , p53 , rat sarcoma virus oncogene ( ras ) , wingless - type ( wnt ) and transforming growth factor ( tgf)- pathways have been frequently found to be genetically or epigenetically altered in hcc .
the cell membrane receptor tyrosine kinase and the downstream ras - mitogen - activated protein kinase and phosphatidyl inositol 3-kinase ( pi3k)-akt kinase signalling pathways are activated by several growth factors whose role in chronic liver diseases and hcc development is established . by binding to their cell surface receptors , fibroblast growth factor ( fgf ) , hepatocyte growth factor ( hgf ) , platelet - derived growth factor ( pdgf ) , tgf- , epidermal growth factor
( egf ) and vascular - endothelial growth factor ( vegf ) activate these pathways and ultimately drive cell proliferation and promote cell survival , but also trigger angiogenesis , invasion and metastasis .
overexpression of met proto - onco - gene ( met ) , the hgf receptor , was found in 4070% of hccs [ 710 ] .
overexpression of vegf was also detected [ 11 , 12 ] and linked to advanced cancer phenotype [ 1317 ] .
together with vegf , overexpression of fgf was associated with angiogenic and invasive phenotypes [ 1820 ] .
overexpression of pdgf and its receptor have also been associated with hcc pathogenesis [ 21 , 22 ] . among downstream effectors ,
overexpression of ras has been demonstrated in hcc , while the presence of activating point mutations was infrequently detected [ 24 , 25 ] .
its activation leads to -catenin stabilization , enabling its translocation to the nucleus , where it interacts with t - cell - specific transcription factor ( tcf / lef ) transcription factors , which , in turn , activate the transcription of target genes including c - myc , c - met , cyclin d1 , vegf , metalloproteases and others .
the wnt--catenin pathway was found to be abnormally activated through several mechanisms in hcc : gain of function mutations at the -catenin n - terminus in 1226% of hccs , deletions or mutations or epigenetic alterations of e - cadherin gene , loss of function mutations at the axin1 or axin2 genes in 813% of hccs [ 27 , 28 ] .
in addition to these mutation events , the increased stability of -catenin may be related to phosphorylation and consequent inactivation of gsk-3 by growth factors , among which insulin , insulin - like growth factor ( igf)-1 , fgf-2 , egf , pdgf , hgf , tgf- and tumour necrosis factor ( tnf)-. a stabilization of -catenin may also result from erk - primed inactivation of gsk-3 or the action of hbv - x protein .
recently , mutations of the -catenin gene have been described in liver adenomas with a higher risk of malignant transformation .
cell proliferation and survival are main outcomes of signal transduction pathways , but several other downstream effectors are also aberrant in cancer .
for example , rb1 , cyclin - dependent kinases ( cdks ) , cyclins and cdk inhibitors , which directly act coordinately to regulate cell cycle , have been extensively examined in hcc . in hcc ,
inactivation of the rb1 gene may occur through either loss of chromosome 13 , found in about 30% of hccs [ 31 , 32 ] , or epigenetic mechanisms , while point mutations are rare .
however , rb1 protein inactivation may derive from the aberrant expression of gankirin , a protein able to bind rb1 and increase rb1 phosphorylation and its degradation by the protea - some .
furthermore , among cell cycle controlling elements , cyclins were reported to be up - regulated , while cell cycle negative regulators were often decreased in hcc tissue , compared with surrounding parenchyma .
cyclind1/cdk4 is overexpressed in about 60% of hccs . among cdk inhibitors , p16/lnik4a , which specifically targets cdk4 and
cdk6 thus inactivating cdk4/cyclin d1 complexes , is functionally inactivated in a large fraction of hccs due to deletions at the short arm of chromosome 9 in about 20% of hccs [ 27 , 31 , 32 ] and methylation of p16/ink4a promoter in 3070% of cases [ 37 , 38 ] .
two other proteins acting as tumour suppressor genes in hcc and directly regulating the cell cycle progression are the cyclin - dependent kinase inhibitors of the cdk interacting protein ( kip ) family cdkn1b / p27 and cdkn1c / p57 .
cdkn1b / p27 and cdkn1c / p57 were reported to be down - regulated in hcc tissue in comparison with surrounding cirrhosis .
we and other groups have previously reported that about 20 to 50% of hccs exhibit a loss of maternal allele methylation at the kvdmri imprinted locus at 11 p15.5 , where cdkn1c / p57 gene is located , and this mechanism has been linked to reduction of cdkn1c / p57 expression [ 4043 ] . as it will be described below , one of the major mechanisms involved in the down - regulation of p27 and p57
is now believed to be the overexpression of the micrornas ( mirnas ) mir-221/222 , which occurs in about 70% of hccs [ 4446 ] . on the contrary , some hccs exhibit high cell proliferation in spite of high p27 expression . in these cases
abnormal cell cycle control is not only a critical step in hcc maintenance , but it also appears to be a critical early event . indeed , an increased proliferation rate and proliferating cell fraction characterize liver cirrhosis too , and correlate with a higher propensity to develop hcc . the contribution of anti - apoptotic factors to hepatocarcinogen - esis has been extensively studied [ 4850 ] . in normal hepatocytes ,
interestingly , trail displays anticancer activity without hepatotoxicity , thus it has been proposed as a possible therapeutic strategy for hcc , which may be further improved by inhibition of proteasome , which was shown to increase susceptibility of hcc cells , but not primary hepatocytes , to trail - induced apoptosis .
the intrinsic pathway relies on the mitochondrial - mediated regulation and it is regulated by the bcl-2 gene family . although bcl-2 expression was not detected in hcc immunohisto - chemical studies , bcl - xl was expressed in all hcc - derived cell lines analysed and in most hcc tissues ; furthermore , its over - expression could independently predict a decreased overall- and disease - free survival [ 54 , 55 ] . among the pro - apoptotic members of the bcl-2 family , a down - regulation of the pro - apoptotic genes bax and bcl - xs was observed in 80% and 64% , respectively , of hccs [ 56 , 57 ] .
similarly , the pro - apoptotic bid was also found down - regulated in all hccs from hbv - infected patients , and its down - regulation was related to hbv - x protein action . at the cross - road between cell cycle and apoptosis is the p53 molecular pathway .
genetic alterations of the tp53 gene have been extensively described in hcc , and has been shown to vary according to different geographic areas , possibly reflecting different etiologic mechanisms .
the g to t transversion of codon 249 of tp53 gene is the typical alteration found in about 50% of hccs consequent to aflatoxin b1 exposure .
conversely , mutations of tp53 unrelated to aflatoxin b1 exhibit a lower frequency ( 1030% ) and are more frequent in advanced cases .
in addition to tp53 mutations , several other factors involved in hepatocarcinogenesis have been reported to impair p53 function .
in particular , interactions between p53 and hbv - x protein lead to a reduced p53-sequence - specific dna binding activity , thus reducing p53 transcriptional activity and blocking p53-mediated apoptosis .
concerning hcv , a modulation of p53 promoter transcriptional activity was demonstrated in hcc - derived cell lines and was ascribed to hcv - core protein .
advanced tumour features include the ability of cancer cells to promote uncontrolled angiogenesis and invade tissues and blood vessels . in this regard , hcc is one of the most vascular solid tumours with high propensity for vascular invasion .
angiogenesis plays a crucial role in hcc development , growth and metastasis and is used as a diagnostic criterion . the switch to an angiogenic phenotype
is triggered by the activation of genes like ras , inactivation of p53 , or cellular stress factors like hypoxia , reactive oxygen species and nutrient deprivation .
signalling pathways crucial for the angiogenic process include growth - factor - mediated pathways such as vegf and fgf receptor signalling as well as the nitric oxide signalling .
vegf expression is stimulated by hypoxia through the hypoxia inducible factor-1 ( hif-1 ) and the igf-2 and it is produced by both endothelial and tumour cells .
the role of vegf in the development of neovascularization of hcc has been extensively reported in the transition between low - grade dysplastic nodules to high - grade dysplastic nodules to early hcc .
furthermore vegf expression correlates with hcc progression , metastatization , tendency towards portal invasion and higher recurrence rate .
in addition to angiogenesis , another distinguished feature of hcc is its propensity towards vascular and tissue invasion .
one of the signalling pathways conferring invasive potential to hcc cells , is mediated by the hgf / met axis .
hgf is the most potent growth factor for hepatocytes and , by binding to its transmembrane tyrosine kinase receptor , met , it promotes proliferation and regeneration , migration , survival and angiogenesis and it is involved in the control of invasive growth both during tumourigenesis and in embryonic development [ 60 , 61 ] .
cytoplasmic downstream effectors of hgf / met signalling axis include phospholipase c ( plc ) , stats , pi3k , erk-1/2 . among genes targeted by hgf / met signalling , mmps and urokinase - type plasminogen activator are thought to play an important role in cell migration .
as exemplified above , significant and complex cross - talks among the different pathways exist and are involved in different aspects of hcc development and progression .
these cross - talks , largely not understood at the molecular level , could potentially account for the resistance to molecularly targeted drugs , which are able to hit pathways only at one or few sites . in this context
indeed , since the expression of many different proteins is affected by the deregulation of a single mirna , the targeting of even a single deregulated mirna for therapeutic purposes may have an effect on several cancer - associated pathways .
thus , recognizing the deregulated mirnas and their protein - coding gene targets is therefore a relevant task for understanding the molecular basis of liver tumourigenesis and for the development of potentially useful diagnostic and therapeutic tools .
in the recent years , several studies revealed that the expression of mirnas is deregulated in human hcc in comparison with matched non - neoplastic tissue [ 6473 ] .
the list of aberrantly expressed mirnas in hcc identified in various studies is reported in table 2 . among these ,
some mirnas were identified as aberrantly expressed by more than one study ( table 3 ) , thus indicating that irrespective of the employed technological platform or set of samples , these mirnas were the most likely involved in liver tumourigenesis .
in addition and in support of this hypothesis , among aberrantly expressed mirnas in hcc , some were also deregulated in other neoplasms .
for example , the up - regulation of the mir-221/222 was also reported in colon , pancreas , stomach , bladder carcinomas and glioblastomas , or mir-21 was also reported in ovarian , lung , breast carcinomas and glioblastomas , while the down - regulation of mir-199a , mir-200b and mir-214 was reported in ovarian cancer and mir-199b in ovarian and lung cancer . in support of their role in livertumourigenesis , gene targets , like the cyclin - dependent inhibitors p27/cdkn1 b and p57/cdkn1c , or the pi3k antagonist phosphatase and tensin homolog ( pten ) , involved in cell cycle and cell death regulation were experimentally validated targets of mir-221/222 and mir-21 .
mirnas aberrantly expressed in hepatocellular carcinoma ( hcc ) compared to non - tumourous liver tissue detected in at least one report results are summarized from the following references : [ 6573 ] .
mirnas aberrantly expressed in hepatocellular carcinoma ( hcc ) reported by more than one study among the up - regulated mirnas , mir-373 was also identified .
this oncogenic mirna was previously found to be up - regulated in testicular cancer and in breast cancer metastasis and protect cells from activated oncogenes [ 74 , 75 ] .
interestingly , some mirnas that are up - regulated in hcc , such as mir-21 , mir-210 , mir-213 and mir-181 b , were also found to be up - regulated in hypoxic conditions .
thus , aberrant mirna expression in hcc may lead to protection from various stress stimuli . among the down - regulated mirna in hcc , several members of the large let-7 family and the mir-143/145 were found .
although for members of the let-7 family there are some controversial reports , these mirnas emerged as consistently down - regulated in several human cancers [ 7682 ] . among the down - regulated mirnas in hcc is the hepato - specific mir-122 , which accounts for about 70% of the mirna population in the adult liver , where it acts as a key regulator of cholesterol and fatty - acid metabolism .
significantly , the down - regulation of mir-122 was detected in more than 70% of hcc .
it was shown that the level of mir-122 expression increases in the mouse liver throughout development , to reach the maximum just before birth .
thus , the loss of expression of mir-122 of hcc cells may represent either a differentiation reversion or a block to a less differentiated status of liver cells .
there are also indications that the down - regulation of mir-122 may have a more direct role in tumourigenesis through the activation of cyclin g1 .
the role of mirnas either as oncogenes or tumour suppressors in human cancer has been established [ 83 , 84 ] .
various studies have also begun to elucidate the molecular functional links between mirna abnormal expression and the hallmarks of malignant transformation : aberrant cell growth , cell death , differentiation , angiogenesis , invasion and metastasis . here , we summarize the available evidence in hcc .
as previously indicated , met , the tyrosine kinase hgf receptor , is overexpressed in 40 to 70% of hccs and is involved in cell motility , invasion and metastasis .
recent reports indicated that met is post - trascriptionally regulated by mir-199a / a * and mir-1 [ 86 , 87 ] ( fig.1 ) .
members of the mir-199 family emerged in several studies as frequently down - regulated in hcc ( see table 3 ) .
its in vivo significance needs to be confirmed , as the studies were confined to cell lines and the mirna that was active on met was the presumed non - mature mir-199a * strand . in spite of the fact that the aberrant down - regulation of mir-1 did not emerge in several mirna expression studies in hcc , it was recently shown that its expression was silenced in hcc cell lines and primary tumours via promoter methylation ; in addition , mir-1 ectopic expression could induce apoptosis and inhibit cell cycle in hcc cell lines .
thus , the mir-1 silencing in hcc may remove a restriction element that permits met overexpression .
receptor tyrosine kinase ( here indicated hgf / met axis solely ) , ras and pi3k pathways affected by the down - regulated ( downward green arrow ) mir-1 , mir-199a * , let-7 and the up - regulated ( upward red arrow ) mir-21 may lead to cell growth , survival , motility , invasion and metastasis .
the cell cycle progression is mainly affected by the mir-221 ( and mir-222 , not indicated ) up - regulation , which can repress the cdk inhibitors cdkn1 b / p27 and cdkn1 c / p57 .
the activation of tyrosine kinase receptors ( rtks ) initiates a downstream cascade of events that lead cells to proliferate .
crucial elements of this signalling transduction pathway are members of the ras family of oncogenes . a molecular link between mirna deregulation and ras expression has been established .
the 3 untranslated regions ( utrs ) of the kras , nras and hras mrnas contain multiple complementary sites for binding of let-7 members , and forced expression of let-7 in human cancer cells reduces ras protein levels ( fig.1 ) .
since let-7 is generally down - regulated in several human cancers , this mechanism could lead to the activation of the ras pathway . as previously mentioned , it is interesting to note that a frequent overexpression but not point mutations of ras has been reported in hcc .
let-7 has also been shown to repress the high - mobility group at - hook 2 ( hmga2 ) oncogene [ 79 , 80 ] , which encodes for a high - mobility group protein oncogenic in a variety of tumours , including benign mesenchymal tumours and lung cancers .
the effect of let-7 on hmga2 was dependent on multiple target sites in the 3utr .
it was reported that chromosomal translocations associated with human tumours disrupt repression of hmga2 by let-7 mirna , thus demonstrating that disruption of a single mirna - target interaction and loss of mirna - directed repression represent mechanisms for the activation of endogenous proto - oncogenes .
the disrupted repression of hmga2 promotes anchorage - independent growth and the growth - suppressive effect of let-7 on lung cancer cells was rescued by overexpression of the hmga2 orf without a 3'utr .
the link between the down - regulation of let-7 and the simultaneous up - regulation of hmga2 was functionally associated with a cancer stem cell signature , at least in the breast cancer cells skbr3 , by yu et al .. these important findings need to be assessed in hcc too .
the importance of let-7 down - regulation in cancer is further supported by studies bytakamizawa et al . and akao et al.[76 , 81 ] , who showed that let-7 can suppress the growth of a549 lung cancer cells and dld-1 colon cancer cells in vitro .
a direct role of mirnas in controlling cell growth by acting on elements of the cell cycle machinery was provided by studies on mir-221 .
mir-221 , which was recently shown to be induced by mycn and repressed by p53 , emerged as a significantly up - regulated mirna in glioblastoma , pancreatic , hepatocellular kidney , bladder , prostate and thyroid cancer [ 4446 , 73 , 79 , 9094 ] , thus suggesting an oncogenic role in several human neoplasms .
its oncogenic function was substantiated by the discovery of its ability to modulate the expression of the cyclin - dependent kinase inhibitor cdkn1b / p27 , a key controller of cell cycle progression [ 46 , 93 ] .
more recently , another cyclin - dependent kinase inhibitor , cdkn1c / p57 , was also shown to be target of mir-221 , strengthening the role of mir-221 in promoting cell cycle progression ( fig.1 ) . moreover ,
the bh3-only protein bmf was recently proven to be a target of mir-221 ( gramantieri and negrini , manuscript in preparation ) . through this mechanism
anoikis , a form of apoptosis induced by the detachment of anchorage - dependent cells from the surrounding extracellular matrix .
evading anoikis is a critical step in the process of metastasis [ 95 , 96 ] .
taken together , the finding that mir-221 could promote cell cycle progression and protect cells from apoptosis outlines the importance of the aberrant expression of one mirna in cancer , whose action of on various targets could simultaneously affect multiple tumourigenic pathways .
the activation of this pathway leads to the activation of akt kinases , which phosphorylate several protein targets that in turn promote cell survival .
it was shown that pten is a direct target of mir-21 ( fig.1 ) , a mirna that is frequently overexpressed in most types of human cancers .
thus , pten could be repressed by overexpression of mir-21 , which would lead to cell survival through pi3k - akt pathway activation .
mir-21 can also down - regulate the tumour suppressor programmed cell death 4 ( pdcd4 ) [ 98 , 99 ] .
it is up - regulated in senescent fibroblasts and it may inhibit proliferation , possibly through the indirect suppression of cdk1/cdc2 kinase
. moreover , it acts as a negative regulator of intravasation , initial step for cancer cell metastasis .
anti - mir-21-transfected rko cells showed an increase of pdcd4-protein and reduced invasion , while overexpression of mir-21 in colo206f significantly reduced pdcd4-protein amounts and increased invasion .
analyses of primary colorectal cancers revealed that an inverse correlation between mir-21 and pdcd4-protein exists , suggesting that mir-21/pdcd4 interaction may be relevant for invasion / intravasation / metastasis of cancer cells .
mir-21 can also down - regulate the tropomyosin-1 , which suppresses anchorage - independent growth of mcf-7 breast cancer cells , further supporting the oncogenic mir-21 functions .
a link between mir-373 and p53 was proven by a study on mir-372 and mir-373 , which were found to cooperate with oncogenic ras to transform primary human cells .
the study proved that these mirnas could confer protection to oncogene - activated p53 pathway .
it was shown that primary human cells undergo growth arrest and senescence in response to mitogenic signals from oncogenes such as ras , by the activation of the p53 pathway , a response that is reversed by the presence of non - functional p53 .
voorhoeve et al . demonstrated that ectopic expression of mir372/373 was sufficient to allow transformation in the presence of wt p53 .
interestingly , this is a characteristic found in testicular germ cell tumours , where , in contrast with other types of tumours , the mir-372/373 cluster is indeed highly expressed in a generally wt - p53 background , suggesting a role in the development of these tumours .
as previously mentioned , mir-122 is down - regulated in more than 70% of hccs .
later , it was found that cyclin g1 can exert a negative regulation on p53 tumour suppressor gene by recruiting the b subunit of pp2a phosphatase to dephosphorilate and activate mdm-2 , thus leading to p53 degradation [ 105 , 106 ] .
as a result , cyclin g1 overexpression can enhance cancer cell growth and its silencing suppresses cell proliferation [ 107109 ] . in a mouse model , the absence of cyclin g1 was associated with a lower susceptibility to develop liver tumours , which was associated with an increased p53 tumour suppressor activity .
the up - regulation of cyclin g1 consequent to mir-122 down - regulation in human hcc may thus lead to p53 down - regulation and promote tumourigenesis .
mirna expression could also be used to improve our ability to classify hcc and stratify their prognostic risk .
the presently used classifications for hcc are exclusively based on clinical parameters [ 3 , 111116 ] .
tumour status ( number and size of nodules , presence of vascular invasion and extrahepatic spread ) , liver function indicators , presence of portal hypertension and performance status are considered for prognostic purposes and for the choice of treatment . because hcc is heterogeneous both from molecular and clinical perspectives [ 117 , 118 ] ,
the available classifications for hcc could be improved and redefined by the incorporation of molecular data .
for example , met - regulated expression signature defines a subset of human hccs with poor prognosis and aggressive phenotype .
cdkn1b / p27 and cdkn1c / p57 exhibits a relevant prognostic significance in human hcc .
cdkn1b / p27 down - regulation is associated with advanced tumour stage , lower survival and higher recurrence rate of small hcc , higher biological aggressiveness , poor differentiation , portal invasion and high proliferative activity . reduced cdkn1c
/ p57 labelling index was associated with worse outcomes and lower disease - free survival after surgery , suggesting that cdkn1c / p57 down - regulation might contribute to the progression of hcc through modulation of cell growth [ 39 , 122 ] .
overexpression of the anti - apoptotic gene bcl - xl independently predicts a decreased overall- and disease - free survival [ 54 , 55 ] .
high throughput technologies have made possible to explore gene expression patterns on large series and to characterise molecular signatures associated with different aetiologies [ 123125 ] , stage [ 126 , 127 ] , propensity to recurrence [ 128130 ] and prognosis [ 131 , 132 ] .
incorporation of these findings in hcc classification could be valuable in improving our ability to stratify hccs ; however , additional validations are still required before their clinical use is granted . in this context ,
more recently , patterns of mirna expression were found to correlate with bio - pathological and clinical parameters , indicating that mirnas could become useful molecular markers for hcc classification and prognostic stratification . among deregulated mirnas ,
up - regulation of mir-221 was associated with shorter time to recurrence ( gramantieri and negrini , unpublished ) .
this is not unexpected , giving the fact that mir-221 regulates the expression of p27 and p57 , two tumour suppressor proteins whose down - regulation was associated with poor prognostic factors in hcc [ 39 , 120122 ] .
recently , up - regulation of mir-210 , one of the mirna that is induced under hypoxia , was associated with reduced disease - free and overall survival in breast cancer .
since mir-210 is also up - regulated in hcc as well as in a variety of solid cancers [ 135138 ] , its prognostic value may possibly be relevant for other cancers too , including hcc .
the study by ladeiro et al . could identify mirna signatures able to classify liver samples according to histology ( tumour / non - tumour ; benign / malignant ; inflammatory adenomas and focal nodular hyperplasia ) , aetiology ( alcohol consumption or hbv infection ) and cancer gene mutations ( -catenin and hepato - cyte nuclear factor 1a ) .
the study by budhu et al . revealed a 20-mirna signature associated with venous invasion ( table 4 ) .
significantly , the same signature could also correlate with disease - free and overall survival .
the results by budhu et al . may be particularly useful to classify patients with hcc at early stage , which may provide a more rational approach to treatment intervention .
hence , either alone or in combination with other parameters , mirna expression patterns may potentially become useful markers for hcc classification and prognostic risk stratification .
mirna signatures associated with hepatocellular carcinoma ( hcc ) metastasis and survival these data were from budhu et al .
to identify novel therapeutic approaches for the treatment of hcc , targeting genes associated with molecular pathways involved in human tumourigenesis has become the most rational approach . in hcc ,
the use of imatinib mesylate , a platelet - derived growth factor receptor ( pdgfr ) and other tyrosine kinases inhibitor , has been suggested [ 140142 ] ; however , in spite of initial positive results , phase 2 clinical trials did not reveal any significant efficacy .
new molecularly targeted agents with a selective action and few side effects on residual liver function are under investigation : taking advantage of the progresses in understanding the molecular pathogenesis of hcc , kinases inhibitors , anti - angiogenic compounds , mtor inhibitors and other molecularly targeted agents have been and are currently under evaluation in phase 2 and 3 trials . at present , the only phase 3 clinical trial so far completed compared the oral multikinase inhibitor sorafenib , which is known to block the kinase activities of vegfr , pdgfr and raf , with placebo in patients with advanced hcc . treated patients displayed a median overall survival of 10.7 months versus 7.9 months in the placebo group , setting the standards for future investigations [ 147 , 148 ] .
this moderate improvement in therapeutic efficacy and overall survival may represent a significant progress , as most of the studies did not rely upon molecular characterization of the different gene targets prior to inclusion in clinical studies , which might have aid the selection of the best target population for the different treatment options .
the discovery that mirnas play an important role in hepato - carcinogenesis has laid the foundation for their exploitation for molecular therapy .
used anti - fas sirna to protect mice from induced acute liver injury and , similarly , zender et al .
used anti - caspase-8 sirnato protect mice against fas ligand - induced liver injury [ 149 , 150 ] . both of these studies demonstrated a survival benefit using sirna approach without significant side effects .
although several studies have established the potential usefulness of mirna - based therapy in cancer [ 76 , 81 , 151 , 152 ] , up to now there has not been any report of using agents that mimic mirnas in animal or clinical models .
more recently , anti - mirna oligonucleotides ( amos ) have been developed . among mirnas
up - regulated in hcc , transfection of cultured glioblastoma and breast cancer cells with amos anti - mir-21 induced inhibition of mir-21 accompanied by suppression of cell growth , associated with increased apoptosis [ 153 , 154 ] .
mir-21 is overexpressed in colangiocarcinomaand its inhibition by amos increases sensitivity to the chemotherapeutic agent gemcitabine .
either cholesterol - bound oligonucleotides ( antagomirs ) , or lna - modified oligonucleotides ( lna anti - mir ) were also found to be effective in stably suppressing mirna activity in the in vivo condition [ 156 , 157 ] .
in addition , liver appeared to be the organ most efficiently and consistently targeted by intravenous injection of amos .
recently , a study performed in african green monkeys assessed safety and efficacy of the approach .
efficient silencing of mir-122 was achieved by three doses of 10 mg / kg lna - anti - mir , leading to a long - lasting and reversible decrease in total plasma cholesterol without any evidence for associated toxicities or histopathological changes in the liver of the animals .
thus , by proving feasibility , safety and efficacy for the use of amos in a pre - clinical setting , these studies established the basis for their use as therapeutic molecules in clinical trials .
the potentiality for therapeutic implementation of small rnas or amos in clinical practice is enormous .
moreover , as far as treatment of liver and hcc is concerned , the limitations encountered for other organs related to an effective in vivo delivery of the drugs is partly overcome by the current clinical application of techniques able to deliver the drugs directly into the hepatic artery branches .
the discovery of aberrantly expressed mirnas in hcc has helped to reveal novel mechanisms in liver tumourigenesis .
understanding molecular tumourigenesis has established the basis for the development of more rational classification and therapeutic approaches . in hcc , aberrantly expressed mirnas could be associated with bio - pathological and clinical features , making mirna expression a potentially useful tool for hcc classification and prognostic stratification , in particular in early hcc , where the availability of potentially curative approaches requires a more sophisticated diagnostic approach .
finally , by revealing which mirna are up- or down - regulated in liver tumourigenesis , new potential therapeutic targets have been revealed . since
technological advancements have shown that the use of mirnas or amos as potentially therapeutic molecules is feasible and safe , their experimentation may become an important area of clinical investigation in the years to come . | hepatocellular carcinoma ( hcc ) is the third cause of cancer - related death worldwide .
curative options for hcc are limited and exclusively available for patients carrying an early stage hcc . in advanced stages , traditional chemotherapy proved to be only marginally effective or even toxic .
thus , the identification of new treatment options is needed .
new targets for non - conventional treatment will necessarily take advantage of progresses on the molecular pathogenesis of hcc
. micrornas ( mirnas ) are a group of tiny rnas with a fundamental role in the regulation of gene expression . aberrant expression of several mirnas was found to be involved in human hepatocarcinogenesis .
mirna expression signatures were correlated with bio - pathological and clinical features of hcc . in some cases ,
aberrantly expressed mirnas could be linked to cancer - associated pathways , indicating a direct role in liver tumourigenesis .
for example , up - regulation of mir-221 and mir-21 could promote cell cycle progression , reduce cell death and favour angiogenesis and invasion .
these findings suggest that mirnas could become novel molecular targets for hcc treatment .
the demonstration of in vivo efficacy and safety of anti - mirna compounds has opened the way to their use in clinical trials . |
however , the incidence of btc appears to be increasing worldwide over the last few decades , particularly in latin america and east asia ( 1 ) .
btc is currently the sixth leading cause of cancer - related mortality in japan , and patients with btc have a poor prognosis ( 2 ) .
however , the majority of patients are diagnosed at an advanced stage , when curative resection is no longer feasible ; in addition , even in cases where surgery may be performed , there is a significant likelihood of relapse ( 3 ) .
patients with unresectable or recurrent disease appear to be clinical candidates for systemic chemotherapy ( 4,5 ) .
after the abc-02 study reported that gemcitabine / cisplatin ( gc ) combination therapy significantly prolonged median survival time ( mst ) from 8.1 to 11.7 months over gemcitabine monotherapy in patients with advanced btc ( 6 ) , this combination therapy has become the standard treatment for btc worldwide .
recently , kanai et al determined the optimal dose of gc / s-1 ( gcs ) combination therapy for patients with advanced btc in a phase i study ( 7 ) and reported a promising mst of > 15 months in a phase ii study ( 8) .
based on these results , a phase iii randomized trial is underway to demonstrate the superiority of gcs therapy compared with gc in patients with unresectable btc ( umin000014371/nct02182778 ) .
we herein report the case of a patient with unresectable intrahepatic cholangiocarcinoma ( icc ) who underwent conversion surgery and ultimately achieved a pathologically complete response using gcs combination therapy .
a 68-year - old woman was referred to toyonaka municipal hospital with increased levels of biliary enzymes in march , 2015 .
the patient suffered from hypertension and rheumatoid arthritis , which were controlled with oral medication .
a physical examination revealed no abdominal abnormalities , but the laboratory tests revealed abnormal liver function , including an elevated serum alkaline phosphatase level of 839 u / l [ upper limit of normal ( uln ) , 328 u / l ) , a -glutamyltranspeptidase level of 324 u / l ( uln , 64 u / l ) and an alanine aminotransferase level of 82 u / l ( uln , 40 u / l ) . additionally , the serum carcinoembryonic antigen ( cea ) and carbohydrate antigen ( ca ) 19 - 9 levels were 9.8 ng / ml ( uln , 5.0 ng / ml ) and 32,806 u / ml ( uln , 37 u / ml ) , respectively . computed tomography ( ct ) revealed a 3-cm mass in the 1/8 segment of the liver , resulting in dilated bile ducts in both hepatic lobes , enlarged para - aortic and regional lymph nodes , and invasion of the portal vein ( pv ) ( fig .
1a ) . magnetic resonance imaging ( mri ) revealed an intrahepatic mass as a hypointense lesion compared with normal liver tissue in t1-weighted images .
t2-weighted images revealed mild hyperintensity compared with the liver parenchyma . furthermore , diffusion - weighted mri revealed high signal intensity ( fig . 1b ) . positron emission tomography with f - fluoro - d - deoxyglucose ( f - fdg pet)/ct revealed abnormal uptake of the primary tumor and para - aortic lymph nodes , with an suvmax of 5.8 and 2.5 , respectively ( fig .
the patient was diagnosed with unresectable intrahepatic btc with para - aortic and hilar lymph node metastasis [ ct3n1m1 , cstage ivb according to the union for international cancer control classification system ( http://www.uicc.org/sites/main/files/private/tnm_classification_of_malignant_tumours_website_15%20may2011.pdf ) ] .
endoscopic retrograde cholangiopancreatography ( ercp ) revealed a 2-cm irregular stricture of the hilar bile duct with a lobulated tumor ( fig .
the brush cytology specimens revealed atypical cells in a three - dimensional cluster and an isolated pattern strongly suggestive of adenocarcinoma ( fig .
, the patient was diagnosed with unresectable icc with para - aortic lymph node metastasis and , therefore , systemic chemotherapy was considered .
the patient underwent endoscopic biliary drainage using plastic stent placement for the biliary stricture prior to therapy .
endobiliary stents ( 7f 12- and 7-cm at a light angle ) were successfully placed through the narrowed lumen at initial ercp , resulting in successful biliary decompression .
the patient was subsequently enrolled in a phase iii randomized trial ( umin000014371/nct02182778 ) and randomly assigned to receive gcs combination therapy .
/ m on day 1 , and oral s-1 was administered daily at a dose of 80 mg / m on days 17 every 2 weeks .
the patient received 12 cycles of the regimen for 6 months . on the first day of the 8th cycle , the patient presented with grade 3 malaise after receiving chemotherapy .
this adverse effect was , however , manageable and improved within 2 days by fluid replacement therapy .
after the scheduled 12 cycles of the regimen , ct revealed a marked reduction of the primary tumor and metastatic lymph nodes .
f - fdg - pet / ct also revealed diminished abnormal uptake of the primary lesion and para - aortic lymph nodes ( fig .
3b ) . additionally , the serum cea and ca19 - 9 levels decreased to within the normal range ( 1.5 ng / ml and 11 u / ml , respectively ) .
the patient achieved a good partial response to gcs therapy and was allowed to undergo conversion surgery .
therefore , the patient underwent extended right hepatic lobectomy , lymph node dissection and left hepaticojejunostomy .
the resected specimen exhibited almost complete occlusion of the right hepatic duct immediately before the junction ( fig .
histological examination revealed scattered pigmented macrophages in the fibrotic tissue , suggesting that necrotic cells were scavenged from this location .
although atypical epithelia in the bile duct were identified , invasive carcinoma and intraepithelial carcinoma components were not found , even following thorough examination .
there were no viable carcinoma cells in the dissected nodes , but some contained fibrotic foci ( fig .
a patient with unresectable icc at presentation achieved a pathologically complete response after undergoing preoperative gcs combination chemotherapy . at the last follow - up , 9 months after the operation ( september , 2016 ) , the patient remained alive and recurrence - free , without adjuvant therapy .
btc , which originates in the intrahepatic and extrahepatic bile ducts , is a relatively uncommon type of cancer , comprising ~3% of all gastrointestinal malignancies ( 9,10 ) .
the majority of btc patients are diagnosed at an advanced stage due to the lack of abdominal symptoms , and the prognosis is generally poor ( 1,2 ) .
therefore , surgery is the optimal therapeutic approach , although systemic chemotherapy is considered for patients with unresectable btc . in the abc-02 study ,
valle et al reported that gc combination therapy was associated with a significant survival advantage compared with gemcitabine alone , with a overall mst of 11.7 months compared with 8.1 months , respectively ( hazard ratio = 0.64 ; p<0.001 ) ( 6 ) . based on that study , gc combination therapy has been the standard palliative chemotherapy for patients with advanced btc worldwide .
the bt-22 study used the same regimen as the abc-02 study and evaluated the efficacy and safety for patients with advanced btc in a japanese population ; the study revealed that its outcome was similar to that of the abc-02 study , as the mst for gc combination therapy and g alone was 11.2 and 7.7 months , respectively , and the adverse events did not significantly differ between the two groups , although the incidence of hematotoxicity was higher with gc combination therapy compared with g alone ( gc vs. g : leukopenia , 29.3 vs. 19.0% ; neutropenia , 56.1 vs. 38.1% ; thrombocytopenia , 39.0 vs. 7.2% ; and decreased hemoglobin level , 36.6 vs. 16.6% , respectively ) ( 11 ) .
therefore , this indicates that the new regimen exhibited a higher efficacy and fewer adverse events .
s-1 is an oral fluoropyrimidine prodrug that is widely used for various solid tumors ( 1215 ) , and it is approved in japan as a chemotherapeutic agent for btc ( 16 ) .
s-1 monotherapy has shown promising outcomes associated with mild toxicity in btc patients ( 13 ) . additionally , in combination with gemcitabine
, s-1 has also achieved favorable response rates ( 3034% ) and mst ( 11.612.7 months ) ( 14,15 ) . on the basis of these reports ,
kanai et al expected that the addition of s-1 would exert an additive or synergistic effect with gc combination therapy to improve treatment results with respect to efficacy and safety . in a phase i study , the regimen described below had the fewest grade 34 adverse events ( maculopapular rash , vasovagal reaction and anemia ) . based on the incidental rates of adverse events in the phase i study , kanai et al established a recommended dose of the gcs combination therapy , which consisted of intravenous administration of gemcitabine ( 1,000 mg / m ) and cisplatin ( 25 mg / m ) on day 1 and oral administration of s-1 ( 80 mg / m ) on days 17 , every 2 weeks ( 7 )
a phase ii study demonstrated significantly prolonged mst ( 16.2 months , 95% confidence interval 10.222.2 months ) without uncontrollable adverse events compared with that of the abc-02 study ( 11.7 months , hazard ratio = 0.64 ; p<0.001 ) .
interestingly , two patients ( 4% ) were able to achieve curative secondary resection after tumor downstaging following chemotherapy ( 8) . thus , gcs combination therapy should not only be considered to be a standard first - line chemotherapy , but also a conversion surgery or neoadjuvant chemotherapy. on the basis of these findings , a randomized phase iii study has now been launched ( umin000014371/nct02182778 ) .
conversion surgery is radical resection performed for previously unresectable cases that become resectable as a result of regression following chemotherapy ; it should be distinguished from neoadjuvant chemotherapy , although a strict distinction between these two strategies is occasionally clinically difficult , as the definition of unresectable cancer varies among physicians .
conversion surgery or neoadjuvant chemotherapy for unresectable cancers , including gastric , colorectal and pancreatic cancer , has been frequently reported ( 1719 ) .
kim et al demonstrated that clinically curative conversion therapy resulted in the highest survival rate and best prognosis in gastric cancer patients with peritoneal seeding ( cstage iv ) .
the mst of patients undergoing clinically curative conversion therapy and non - curative resection was 37 and 18 months , respectively , and the 3-year survival rates were 50 and 0% , respectively ( 17 ) . for colorectal and pancreatic cancer
, several recent reports also demonstrated a clinical advantage with neoadjuvant chemotherapy ( 18,19 ) .
however , the feasibility and efficacy of neoadjuvant chemotherapy for btc has not been determined ( 20 ) .
kato et al reported that patients with initially unresectable locally advanced btc who underwent neoadjuvant chemotherapy ( gemcitabine ) had a significantly longer survival time compared with those unable to undergo surgery ( 2-year overall survival rate of 45 and 19% , respectively ) ( 21 ) .
furthermore , a case of curative resection after gcs chemotherapy for initially unresectable biliary duct cancer and a case of a patient with extrahepatic cholangiocarcinoma after undergoing preoperative gemcitabine - based chemotherapy have been reported ( 22 ) .
based on these reports , conversion therapy has recently attracted attention , although there is insufficient evidence regarding the safety and efficacy of performing conversion surgery . to the best of our knowledge , the present case is the first report of a patient diagnosed with unresectable btc who ultimately achieved a pathologically complete response with gcs combination therapy .
further studies are required to verify the efficacy of conversion surgery in patients with btc using a prospective study design . | a 68-year - old woman was referred to our hospital with increased levels of biliary enzymes . on imaging ,
the patient was diagnosed with unresectable intrahepatic biliary tract cancer ( btc ) with invasion of the portal vein and para - aortic lymph node metastasis ( ct3n1m1 , cstage ivb ) and underwent endoscopic biliary drainage for the biliary stricture prior to therapy .
the patient was subsequently enrolled in a phase iii randomized trial ( umin000014371/nct02182778 ) and randomly assigned to receive gemcitabine / cisplatin / s-1 ( gcs ) combination therapy intravenously at doses of 1,000 or 25 mg / m2 on day 1 and orally twice daily at a dose of 80 mg / m2 on days 17 every 2 weeks .
after 12 cycles of scheduled therapy without uncontrollable adverse effects , the patient achieved a good partial response with chemotherapy . computed tomography ( ct ) revealed a marked reduction of the primary and metastatic lesions . in addition,18f - fluorodeoxyglucose - positron
emission tomography / ct revealed diminishing abnormal uptake and no macroscopic evidence of factors adversely affecting tumor resectability .
therefore , the patient underwent extended right hepatic lobectomy , lymph node dissection and left hepaticojejunostomy . finally , histological examination of the resected tissues revealed no residual cancer cells , suggesting a pathologically complete response .
we herein present the case of a patient with intrahepatic btc who achieved a pathologically complete response following combination chemotherapy with gcs . |
amyotrophic lateral sclerosis ( als ) is a devastating neurodegenerative disease which affects both motor function and extramotor systems . according to the revised el escorial criteria for the diagnosis of als , the presence of signs for the affection of both upper motor neurons ( umns ) in the primary motor cortex and lower motor neurons ( lmns ) in brainstem and spinal cord is mandatory , and the disease must be progressive . because of varied clinical phenotypes of als , it is difficult to differentiate it from other als - mimicking conditions .
electromyography can be used for the detection of lmn involvement in addition to the clinical examination . on the other hand
, umn signs must be visible at the clinical examination while electrophysiological transcranial motor stimulation abnormalities are not included in making the diagnosis of als according to the el escorial criteria .
magnetic resonance imaging ( mri ) is the radiological investigation of choice with the recent advances of diffusion tensor imaging , presenting a promising technique for early detection of alterations in the motor cortex and pyramidal tracts .
herein , we report a case of a 9-year - old male who presented to the department of neurology with complaints of progressive increase in weakness of bilateral lower limb , poor grip in bilateral upper limb , and difficulty in walking since last 2 years . there was no history of any muscle pain , trauma , fever , recent vaccination , dog bite , respiratory tract or gastrointestinal tract infection .
physical examination demonstrated decreased power in the bilateral lower limb ( 3/5 in the right lower limb and 2/5 in the left lower limb ) and upper limb ( 4/5 in the right hand and 4/5 in the left hand ) , no signs of dermal rashes , or hypertrophy of muscles .
mri of the brain was performed using 1.5 tesla scanner ( ge signa excite , usa ) .
t2-weighted imaging and additional fluid attenuating inversion recovery sequence showed hyperintense signals in the posterior limb of internal capsule [ figure 1a and b ] involving corticospinal tract ( cst ) which was found to extending up to midbrain .
( a ) t1-weighted imaging , black arrow shows hyperintensity in the posterior limb of internal capsule ( b ) t2-weighted imaging , black arrow shows hyperintensity in the posterior limb of bilateral internal capsule ( c ) t2-weighted imaging , black arrow shows hyperintensity in the posterior bilateral internal capsule and midbrain ( corticospinal tract ) with classical wine glass appearance
areas of abnormal signal intensity on t1- and t2-weighted images in the cst have been previously reported in patients with als .
other diseases such as ischemic processes , demyelinating diseases , and infections may involve the pyramidal tracts . however , bilateral symmetrical involvement associated with the characteristic clinical findings clinches the diagnosis of als .
the degeneration of motor neurons may result in a cellular loss and axonal edema as previously shown on electronic microscopy .
so far , imaging methods had been of limited use because of their unsatisfactory results . approximately , 90% of als are sporadic with 10% having familial predisposition , but majority of the cases of juvenile als ( jals ) are familial .
most of the jals are autosomal recessive , though autosomal dominant inheritance patterns have been described .
recessive forms of jals have been mapped to chromosome regions 2q33 and 15q12 - 21 .
no effective treatment for the disease have been found till date , though thyrotropin - releasing hormone , ganglioside therapy , and plasmapheresis have been tried in adults without much benefit .
jals is a diagnosis that should be suspected in a child presenting with a combination of umn and lmn signs .
mri facilitates reaching the correct diagnosis by showing the typical involvement of the cst and the wine glass appearance .
mri plays as an important diagnostic tool for reaching up to a diagnosis and for prognosticating the disease .
| amyotrophic lateral sclerosis ( als ) , also known as lou gehrig disease , is a chronic degenerative neurologic disease and is characterized by the selective involvement of the motor system .
usually , patients present with upper motor neuron ( umn ) and lower motor neuron compromise .
degeneration of the umn in the cerebral cortex is one of the main pathologic changes in als .
these changes usually affect corticospinal tracts leading to degeneration of the fibers which show characteristic hyperintensities along the tracts leading to the wine glass sign .
patients with als usually present in the sixth decade of life ; presentation in pediatric age in the form of juvenile als being rare . |
the valid extrapolation of carcinogenesis data from one species to another depends , in part , on strong similarities of the metabolic and cellular mechanisms involved in the carcinogenic process and similarities in the nature and behavior of the various lesions that appear during the development of neoplasia between the species involved .
although there are many biological differences between the various rodent species used in carcinogenesis research , there are more similarities , in keeping with the surprising unity of basic cellular and tissue organization and function that is evident throughout biological systems at every level of evolutionary development .
an understanding of intraspecies similarities and differences , especially as these modify the morphologic responses of the host to carcinogenic chemicals , is of central importance if carcinogenesis data from one species are to be used to predict carcinogenic risk in another . in this manuscript
the histopathology of the various lesions that appear during chemically induced cancer of the liver , pancreas , and bladder in several rodent species has been selected to compare and contrast similarities and differences that exist among them and among the spontaneous premalignant lesions and carcinomas of these organs in humans.imagesfigure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12.figure 13.figure 14.figure 15 . | |
a 34-year - old man with painless right scrotal swelling , first noticed four months earlier , was admitted to our hospital .
the patient had no history of scrotal trauma , voiding difficulty , other genitourinary diseases or weight loss .
physical examination showed that the right side of the scrotum was noticeably enlarged , without tenderness .
laboratory studies were unremarkable , with normal levels of alpha - fetoprotein ( 1.70 ng / ml ; normal range : < 20 ng / ml ) and beta - human chorionic gonadotropin ( 3.42 miu / nl ; normal range : < 5 miu / nl ) . under the impression of right epididymo - orchitis ,
scrotal us revealed a well - defined heterogeneous hypoechoic intratesticular lesion of the right testis , with large hydrocele and diffuse irregular epididymal swelling .
also noted were slightly heterogeneous echogenic nodular projections attached to the inner scrotal wall ( fig .
for further evaluation , mr imaging was performed , and this demonstrated nodules in the testis , epididymis and inner scrotal wall .
the nodules were isointense on t1-weighted images and slightly hyperintense on t2-weighted images . t2-weighted imaging of the intratesticular lesion revealed a low - signal - intensity rim .
after infusion of gadopentetate dimeglumine ( gd ) ( magnevist ; berlex , wayne , n.j .
, u.s.a . ) , the peripheral portions of these nodules were markedly enhanced ( fig . 1b - e ) . because malignancy was suspected , the patient underwent right radical orchiectomy , and right hydrocele with testis was dissected out without difficulty . on pathologic examination of the resected specimen ,
variable - sized multiple nodules were seen on the surface of the tunica vaginalis and albuginea ; the largest one was attached to the posterolateral portion of the testis .
microscopic examination revealed that the nodules were confined to the tunica vaginalis and albuginea , without penetrating the testis , and between this and the nodules there was a clear plane of separation ( fig .
the tumor cells were oval , or round to elongated , with occasional longitudinal nuclear grooves and call - exner bodies , and mitosis was frequently observed ( nine mitoses / ten high - power fields , on average ) .
a comparison of the microscopic findings and those of mr imaging indicated that the hypointense rim seen at the periphery of the nodules on t2-weighted images corresponded with intact tunica albuginea .
although granulosa cell tumor is the most common sexcord stromal tumor to occur in the ovary , it is extremely rare in the testis and its origin is poorly understood . to the best of our knowledge ,
the literature in english is limited to only 19 cases of adult granulosa cell tumor of the testis ( 10 ) .
we are aware of only two previous reports of the us findings of adult - type testicular granulosa cell tumor , and both of these described the tumor as a well - defined hypoechoic testicular mass ( 11 , 12 ) . in our case , the tumor appeared to arise from the scrotal tunics , an unusual location for a sex - cord stromal tumor .
it was , however , because of the presence of typical large indented nuclei with longitudinal nuclear grooves and call - exner bodies , as well as positive immunoreactivity for inhibin , keratin and vimentin , that granulosa cell tumor was diagnosed ( 13 ) .
fourteen percent of all paratesticular tumors arise from the scrotal tunics and among these , benign fibroma , representing a fibrous reaction to chronic periorchitis , is the most common lesion ( 14 ) .
although granulosa cell tumor arising from the scrotal tunics has not been previously reported , extratesticular sex - cord stromal tumor in the pelvis has been described ( 9 ) .
the suggested explanation is that the tumor originated in a residual part of the primitive gonad or a third undescended testis which was completely destroyed by the process , and we believe that in our case the same explanation is possible . pathologic examination indicated that this tumor had originated from and was confined to the scrotal tunics .
nevertheless , because the largest nodule was deeply invaginated into the testis , displacing the epididymis , which was compressed and not visualized , the location of this tumor - according to the findings of us and mr imaging - was intratesticular and epididymal .
it was because the main lesion was in the testis and epididymis , and multiple small nodules were attached to the inner scrotal wall , that malignancy was suspected . on the other hand , because of extensive hydrocele , the lesion also mimicked an inflammatory condition such as tuberculous epididymo - orchitis .
the only clue to the origin of the mass was the low - signal - intensity rim between the lesion and the testis revealed by t2-weighted imaging .
to our knowledge , this is the first reported case of paratesticular granulosa cell tumor , and it would therefore be premature to state that the us and mr features of our case are specific to granulosa cell tumor of the scrotal tunics . although the formulation of a treatment plan might not require specific diagnosis , we suggest that the presence of a mass seen as an intratesticular lesion associated with multiple enhancing nodules attached to scrotal tunics indicates the possibility of a paratesticular tumor , and that granulosa cell tumor should be included in the differential diagnosis .
we believe that mr imaging findings of a peripheral low - signal - intensity rim on a t2-weighted image might help determine the origin of the mass . | we report a case of adult granulosa cell tumor arising in the scrotal tunics . the patient was a 34-year - old man who presented with right scrotal swelling , first noticed four months previously . under the initial clinical impression of epididymoorchitis ,
antibiotic treatment was instituted but there was no response .
the paratesticular nodules revealed by ultrasound and magnetic resonance imaging mimicked intratesticular lesion , and radical orchiectomy was performed .
although several cases of adult testicular granulosa cell tumor , have been reported , the occurrence of this entity in the paratesticular area has not , as far as we are aware , been previously described . |
as the most common aneurysm , abdominal aortic aneurysm ( aaa ) mainly occurs in middle - aged or elderly people , and shows an increasing trend worldwide . with the transition of demographic distribution toward an aging society , the incidence of aaa in china is also increasing .
although having a benign nature , aaa still has relatively high mortality due to rapid progression and eventual rupture without timely diagnosis and intervention .
therefore , understanding the pathogenesis of aaa can greatly benefit screening in high - risk populations for making timely diagnosis and treatment , and thus has become a research focus in recent years .
the etiology of aaa involves a complicated process containing a cascade reaction with multiple factors and steps , including genetic / environmental factors and abnormal anatomy , and alternation of hemodynamics and inflammatory response [ 46 ] . among all those risk factors ,
atp - binding cassette transport protein a1 ( abca1 ) is the most important cell - surface protein facilitating efflux of cholesterol identified to date .
it exerts its function mainly through the regulation of reverse cholesterol transport ( rct ) , thus mediating the level of high - density lipoprotein ( hdl ) . in patients with either tangier disease or hdl deficiency , mutations of abca1 gene
have been identified , causing deficits of apolipoprotein a - i ( apoa - i ) and hdl , leading to the insufficiency of serum hdl and further atherosclerosis .
genetic studies have found the prominent distribution of abca1 mutations in the binding domain and n - terminal region of this gene . in certain atherosclerosis patients , single - nucleotide polymorphism ( snp ) of abca1 gene
other studies have also confirmed the correlation between snp of abca1 gene and both atherosclerosis and lipid metabolic disorders .
therefore , this study aimed to investigate the correlation between abca1 gene polymorphism of aaa patients and apoa - i and hdl - c , in an attempt to analyze its relationship with aaa pathogenesis .
a total of 126 aaa patients ( including 86 males and 40 females , ages 12~89 years , average age=62.312.7 years old ) who were diagnosed and treated in our hospital between january 2012 and december 2014 were recruited in this study .
all patients received confirmed diagnosis of aaa via abdominal ultrasound , doppler ultrasound , mscta , or dsa .
we recruited 119 healthy volunteers from routine health check - ups in our hospital . among the control group ,
there were 72 males and 47 females , ages 12~78 years ( average age=61.514.1 years old ) .
control group individuals had no atherosclerosis and had normal blood sugar / lipid levels as determined by ultrasound and laboratory tests .
any medications that potentially affect lipid metabolism were suspended for 2 weeks before collecting the blood samples .
this study was pre - approved by the ethics committee of our hospital and we obtained written consents from all participants or their guardians
. fasted venous blood samples ( 10 ml ) were collected within 24 h after admission from elbow vein puncture on all patients .
generation information of participants , including age , sex , and body - mass index ( bmi ) , were recorded along with other risk factors , including blood pressure and smoking habits .
an automatic biochemical analyzer was used to detect biochemical indexes , including triglyceride ( tg ) , hdl - c , total cholesterol ( tc ) , and low - density lipoprotein ( ldl - c ) by standard enzymatic assays .
genomic dna was extracted from peripheral erythrocytes using a whole - blood genomic dna extraction kit ( shanghai shenggong bioengineering technology , china ) following the manual instructions . in brief , 1
after centrifugation at 12 000 rpm for 11min and discarding the supernatant , 0.2 ml solution a ( pbs buffer ) was added in the precipitation for re - suspension , followed by the addition of 20 l rnase a ( 10 mg / ml ) .
the mixture was then incubated at room temperature for 10 min , followed by the addition of 20 l proteinase k ( 10 mg / ml ) .
the sample was then resolved by 65c water bath and loaded onto the absorbent column , which was centrifuged and eluted by elution buffer .
the pcr reaction mixture included specific primers ( 0.1 m ; forward , 5-gtatt tttgc aaggc tacca gttac atttg acaa-3 ; reverse , 5-gattg gcttc aggat gtcca tgttg gaa-3 ; designed as previously documented and synthesized by shanghai shenggong bioengineering technology , china ) , 0.5 g of genomic dna , 0.25 mm dntps , tag polymerase , 2 mm mgcl2 and 10x tag buffer ( invitrogen , us ) .
the amplification parameters were : 94c per - denature for 5 min , followed by 30 cycles each consisting of 94c denature for 1 min , 58c annealing for 50 s and 72c elongation for 25 s. the reaction ended with 72c elongation for another 10 min .
pcr products ( 10 l ) were digested by 2 l restriction enzyme ( takara , china ) at 37c for 6 h. the digestion products were further amplified and sequenced by shanghai shenggong bioengineering technology , china .
distribution of alleles and genotypes at loci rs2230806 ( r219k ) of abca1 gene was determined by sequencing .
spss 20.0 software package was used to analyze all collected data , of which enumeration data , including genotype and allele frequencies , were analyzed by chi - square test .
the homogeneity of the sample was further evaluated by hardy - weinberg ( h - w ) equilibrium .
one - way analysis of variance ( anova ) was used to compare means among multiple groups . the correlation between genotype / allele frequency and occurrence of aaa
a total of 126 aaa patients ( including 86 males and 40 females , ages 12~89 years , average age=62.312.7 years old ) who were diagnosed and treated in our hospital between january 2012 and december 2014 were recruited in this study .
all patients received confirmed diagnosis of aaa via abdominal ultrasound , doppler ultrasound , mscta , or dsa .
we recruited 119 healthy volunteers from routine health check - ups in our hospital . among the control group ,
there were 72 males and 47 females , ages 12~78 years ( average age=61.514.1 years old ) .
control group individuals had no atherosclerosis and had normal blood sugar / lipid levels as determined by ultrasound and laboratory tests .
any medications that potentially affect lipid metabolism were suspended for 2 weeks before collecting the blood samples .
this study was pre - approved by the ethics committee of our hospital and we obtained written consents from all participants or their guardians .
fasted venous blood samples ( 10 ml ) were collected within 24 h after admission from elbow vein puncture on all patients .
generation information of participants , including age , sex , and body - mass index ( bmi ) , were recorded along with other risk factors , including blood pressure and smoking habits .
an automatic biochemical analyzer was used to detect biochemical indexes , including triglyceride ( tg ) , hdl - c , total cholesterol ( tc ) , and low - density lipoprotein ( ldl - c ) by standard enzymatic assays .
genomic dna was extracted from peripheral erythrocytes using a whole - blood genomic dna extraction kit ( shanghai shenggong bioengineering technology , china ) following the manual instructions . in brief , 1
after centrifugation at 12 000 rpm for 11min and discarding the supernatant , 0.2 ml solution a ( pbs buffer ) was added in the precipitation for re - suspension , followed by the addition of 20 l rnase a ( 10 mg / ml ) .
the mixture was then incubated at room temperature for 10 min , followed by the addition of 20 l proteinase k ( 10 mg / ml ) .
the sample was then resolved by 65c water bath and loaded onto the absorbent column , which was centrifuged and eluted by elution buffer .
the pcr reaction mixture included specific primers ( 0.1 m ; forward , 5-gtatt tttgc aaggc tacca gttac atttg acaa-3 ; reverse , 5-gattg gcttc aggat gtcca tgttg gaa-3 ; designed as previously documented and synthesized by shanghai shenggong bioengineering technology , china ) , 0.5 g of genomic dna , 0.25 mm dntps , tag polymerase , 2 mm mgcl2 and 10x tag buffer ( invitrogen , us ) .
the amplification parameters were : 94c per - denature for 5 min , followed by 30 cycles each consisting of 94c denature for 1 min , 58c annealing for 50 s and 72c elongation for 25 s. the reaction ended with 72c elongation for another 10 min .
pcr products ( 10 l ) were digested by 2 l restriction enzyme ( takara , china ) at 37c for 6 h. the digestion products were further amplified and sequenced by shanghai shenggong bioengineering technology , china .
distribution of alleles and genotypes at loci rs2230806 ( r219k ) of abca1 gene was determined by sequencing .
spss 20.0 software package was used to analyze all collected data , of which enumeration data , including genotype and allele frequencies , were analyzed by chi - square test .
the homogeneity of the sample was further evaluated by hardy - weinberg ( h - w ) equilibrium .
one - way analysis of variance ( anova ) was used to compare means among multiple groups . the correlation between genotype / allele frequency and occurrence of aaa
we compared general information , including age , sex , smoking history , diabetes , and hypertension , between disease and control groups . no statistically significant difference regarding sex or age
results ( table 1 ) showed significantly higher incidence of hypertension in aaa patients compared to the control group .
the male - to - female ratio of aaa incidence was 2.15-fold higher ( p<0.05 ) .
other indexes , including bmi , smoking , diabetes , and fasted blood glucose ( fbg ) level , showed no significant difference between the 2 groups ( p>0.05 ) .
as shown in table 2 , aaa patients had significantly higher tg and ldl - c levels compared to those in the control group , along with lower hdl - c and apoa - i levels ( p<0.05 in all cases ) .
the distribution of genotypes in both groups fit hardy - weinberg equilibrium , suggesting the homogeneity of both populations . in a comparison of polymorphisms at loci rs2230806 ( r219k ) of abca1 gene ,
the frequency of kk and rr genotypes was significantly different between the disease and control groups ( table 3 , p<0.05 ) .
. a further analysis of allele frequency showed significantly lower incidence of r allele in the disease group compared to the control group ( p<0.05 ) .
a further analysis of the correlation between abca1 genotype and blood lipid level showed significantly higher apoa - i and hdl - c levels in aaa patients carrying r alleles ( both rr and rk genotypes at r219k loci ) .
the level of tc , tg , and ldl - c , however , showed no statistically significant difference between the 2 groups ( table 4 , p>0.05 ) .
logistic regression analysis was used to analyze the correlation between abca1 genotypes and the occurrence of aaa .
as shown in table 5 , no significant correlation was discovered between kk genotype and aaa occurrence .
rr genotype , however , was correlated with aaa occurrence ( or=0.45 , p<0.05 ) , as was rk genotype ( or=0.56 , p<0.05 ) .
the r allele , therefore was correlated with aaa pathogenesis ( or=0.66 , p<0.05 ) .
we compared general information , including age , sex , smoking history , diabetes , and hypertension , between disease and control groups . no statistically significant difference regarding sex or age
results ( table 1 ) showed significantly higher incidence of hypertension in aaa patients compared to the control group .
the male - to - female ratio of aaa incidence was 2.15-fold higher ( p<0.05 ) .
other indexes , including bmi , smoking , diabetes , and fasted blood glucose ( fbg ) level , showed no significant difference between the 2 groups ( p>0.05 ) .
as shown in table 2 , aaa patients had significantly higher tg and ldl - c levels compared to those in the control group , along with lower hdl - c and apoa - i levels ( p<0.05 in all cases ) .
the distribution of genotypes in both groups fit hardy - weinberg equilibrium , suggesting the homogeneity of both populations . in a comparison of polymorphisms at loci rs2230806 ( r219k ) of abca1 gene ,
the frequency of kk and rr genotypes was significantly different between the disease and control groups ( table 3 , p<0.05 ) .
. a further analysis of allele frequency showed significantly lower incidence of r allele in the disease group compared to the control group ( p<0.05 ) .
a further analysis of the correlation between abca1 genotype and blood lipid level showed significantly higher apoa - i and hdl - c levels in aaa patients carrying r alleles ( both rr and rk genotypes at r219k loci ) .
the level of tc , tg , and ldl - c , however , showed no statistically significant difference between the 2 groups ( table 4 , p>0.05 ) .
logistic regression analysis was used to analyze the correlation between abca1 genotypes and the occurrence of aaa . as shown in table 5 , no significant correlation was discovered between kk genotype and aaa occurrence .
rr genotype , however , was correlated with aaa occurrence ( or=0.45 , p<0.05 ) , as was rk genotype ( or=0.56 , p<0.05 ) .
the r allele , therefore was correlated with aaa pathogenesis ( or=0.66 , p<0.05 ) .
as one of the most common dilated vascular diseases , aaa can be classified as symptomatic and asymptomatic sub - types .
the former has relatively high diagnostic rate and treatment efficacy , but the asymptomatic aaa presents an insidious disease onset , impeding timely treatment and causing a higher mortality rate after aneurysm rupture and hemorrhagic shock . therefore , both etiology and treatment of aaa are now receiving considerable research interest . under influences from various predisposing factors , aaa may weaken the basal layer of the aortic wall , causing elevated degradation and decreased synthesis of elastin .
the hemodynamic force further expands the aneurysm , aggravating the disease condition due to atherosclerosis and vascular dilation . in previous studies ,
the present study revealed the male - biased occurrence of aaa , which is consistent with previous reports .
a significantly higher percentage of hypertension existed in the disease group when compared to the control group , suggesting the relationship between hypertensive atherosclerosis and aaa occurrence .
other studies reported smoking , obesity , and abnormal blood glucose as factors affecting aaa .
this study , however , did not find any significant difference regarding bmi , smoking , or diabetes between aaa patients and control individuals .
abnormal blood lipid level usually induces atherosclerosis , which further facilitates the occurrence of aaa .
this study found significantly elevated blood tg and ldl - c in aaa patients , as well as lower hdl - c and apoa - i levels .
as the most important gene regulating human lipid metabolism , abca1 gene locates at 9q31 region and contains more than 50 exons plus 49 introns , making the total length of the gene over 150 kb .
the promoter region , which is localized 1453 bp upstream of the start codon , has multiple binding sites for transcriptional factors involved in lipid metabolism regulation .
abca1 gene has been suggested to be related with atherosclerosis and lipid metabolic disorders , both of which contribute to the occurrence and progression of aaa .
the snp pattern of abca1 has been reported , especially at r219k loci , which locates at 1051 nt of 7 exon , which has significant relationship with various diseases , including hyperlipidemia , coronary heart disease , and atherosclerosis , further suggesting the important role of abca1 gene polymorphism in aaa .
this study revealed a statistically significant difference in frequency of kk and rr genotypes between aaa patients and the control group , as supported by lower incidence of rr genotype and r alleles in the disease group .
further correlation analysis showed higher apoa - i and hdl - c levels in r allele carriers within aaa patients compared to non - carrier patients .
moreover , both rr / rk genotypes and r allele were closely correlated with aaa pathogenesis .
this study for the first time demonstrates the close relationship between r219k polymorphism of abca1 gene and occurrence of aaa ; r allele at this locus may regulate the pathogenesis and progression of aaa .
therefore , further studies are needed on the detailed mechanism of r219k mutation - induced blood lipid abnormalities .
such studies are expected to provide evidence useful for developing large - scale screening , early diagnosis and treatment of aaa . | backgroundas the most common type of aneurysm , abdominal aortic aneurysm ( aaa ) has an unfavorable prognosis due to the high frequency of rupture .
studies have indicated a close relationship between the pathogenesis and progression of aaa and abnormal serum lipid levels .
atp - binding cassette transport protein a1 ( abca1 ) is a cell - surface protein facilitating cellular efflux of cholesterol .
the single - nucleotide polymorphism ( snp ) of abca1 gene has been suggested to be correlated with abnormal metabolism of lipids .
therefore , this study aimed to investigate the relationship between abca1 polymorphism and apoa - i and hdl - c in an attempt to elucidate its correlation with aaa occurrence.material/methodswe included 126 aaa patients and 119 healthy controls in this study .
pcr and restriction fragment length polymorphism ( rflp ) were used to detect the snp pattern of abca1 gene at locus rs2230806 from both aaa patients and healthy controls . the distribution pattern and correlation with
apoa - i and hdl - c was analyzed.resultsthe distribution of kk / rr genotype of abca1 gene had significant difference between disease and control group , with lower rates of rr genotype and r allele in the disease group ( p<0.05 ) .
levels of apoa - i and hdl - c , but not triglyceride and ldl - c levels , in aaa patients who carried r allele in abca1 gene ( including rr and rk genotypes ) were higher than in non - carriers ( p<0.05 ) .
the r allele of abca1 gene was shown to be related with the occurrence of aaa ( p<0.05).conclusionspolymorphism of abca1 gene is correlated with aaa occurrence , possibly via the regulation of serum lipid metabolism by r allele . |
fibrosarcoma is a tumor of mesenchymal cell origin that is composed of malignant fibroblasts in a collagenous background .
it can occur as a soft tissue mass or as a primary or secondary bone tumor .
the sarcomas as a group differ from malignant epithelial neoplasms by their typical occurrence in relatively younger persons .
it is central , arising within the medullary canal or peripheral , arising from the periosteum .
secondary fibrosarcoma of bone arises from a preexisting lesion or after radiotherapy to an area of bone or soft tissue .
several inherited syndromes such as multiple neurofibromas may have a 10% risk over a lifetime of developing a malignant peripheral nerve sheath tumor or fibrosarcoma .
fibrosarcoma also has been noted to arise from preexisting lesion such as fibrous dysplasia , chronic osteomyelitis , bone infarcts , paget 's disease and in previously irradiated areas of bone .
clinically , fibrosarcomas most often present as slow - growing masses that may reach considerable size before they produce pain .
histologically , well - differentiated fibrosarcomas consist of fascicles of spindle - shaped cells that classically form a herringbone pattern .
the cells often show little variation in size and shape although variable numbers of mitotic figures can usually be identified . in poorly differentiated tumors ,
the histological appearance of high - grade fibrosarcoma may be similar to other tumors such as malignant fibrous histiocytoma , liposarcoma or synovial sarcoma .
the positive immunostaining for vimentin , together with negativity for muscular immunomarkers , helps in diagnosing fibrosarcoma .
the treatment of choice is radical surgery ; radiation therapy and chemotherapy can be used in inoperable cases .
a 22-year - old female came with the chief complaint of swelling over the left front side of the face since 4 months [ figure 1 ] .
the patient gave a history of rapidly enlarging swelling which attained the present size of 3 cm 4 cm .
extraorally , the swelling extended superoinferiorly from infraorbital margin to the upper lip and anteroposteriorly from midline to the corner of the mouth .
intraorally , the swelling appeared to be arising from the labial vestibule extending up to the second premolar of the left side [ figure 2 ] .
the buccal cortical plates were expanded but intact . there was displacement and grade ii mobility in relation to 21 , 22 and 24 .
extraorally , a swelling was noticed on the left side of face intraorally , the swelling was seen involving the left canine and extending up to the second premolar the orthopantomography revealed unilocular radiolucency associated with 11 , 12 , 21 , 22 , 24 and root resorption in relation to 21 , 22 , 24 with missing 23 [ figure 3 ] .
radiograph showing well - defined radiolucency and resorption of teeth informed consent was taken from the patient and surgical excision was done and the tissue was sent for the histopathological examination .
the hematoxylin and eosin stained sections revealed the presence of parakeratinized stratified squamous epithelium overlying the connective tissue stroma .
the cells were arranged in fascicular pattern with few areas showing herringbone pattern of arrangement [ figure 4 ] .
the cells were dysplastic in nature with most of them showing cellular and nuclear pleomorphism .
the lesional cells were separated from the overlying epithelium by a zone of connective tissue . [ figure 7 ] . under high power
the typical herring bone pattern was seen [ figure 8 ] . based on the histological assessment , the final diagnosis of intermediate grade fibrosarcoma was made .
photomicrograph showing the spindle cells arranged in herringbone pattern ( h&e stain , 40 ) lesional cells showing positivity for vimentin ( ihc stain , 200 ) few areas showing round cells ( h & e stain , 400 ) the lesional cells were separated from the overlying epithelium by a zone of connective tissue ( h&e stain , 40 ) dysplastic spindle cells arranged in herringbone pattern ( h&e stain , 200 )
this is a type of sarcoma that is predominantly found in the area around the bones or in soft tissue . in earlier studies of soft tissue neoplasm
, this tumor has been greatly overdiagnosed and this diagnosis has been frequently applied to virtually any richly cellular , collagen - forming spindle - cell tumor including malignant fibrous histiocytoma , malignant peripheral nerve sheath tumor and a host of other sarcomatous and pseudosarcomatous lesions .
it is a rare tumor , accounting for approximately 5% of all malignant intraosseous tumors and especially affects the long bones .
of all the fibrosarcomas occurring in humans , only 0.05% occur in the head and neck region . of this , almost 23% is seen in the oral cavity .
fibrosarcoma may arise as a primary tumor in any part of the jaws and may be classified as either peripheral ( periosteal ) or central ( endosteal ) type .
secondary fibrosarcoma of the bone may be associated with fibrous dysplasia , paget 's disease , bone infarct or cyst and/or osteomyelitis ; it may also occur as a malignant transformation of giant - cell tumor of the bone or be induced by prior irradiation .
clinically , in the oral cavity , the major symptoms are pain , swelling and sometimes loosening of the teeth and paresthesia .
radiographically , an osteolytic lesion is usually present , with ill - defined borders ; however , fibrosarcoma of the jaws can not be distinguished from other destructive lesions of the bone .
the cells are rather uniform and spindle - shaped and arranged in fascicles , often forming a herringbone pattern .
histologically , the degree of differentiation of this neoplasm is variable , comparable to either a benign fibroma or an anaplastic tumor .
therefore , care should be taken to distinguish it from other spindle - cell neoplasms .
fibrosarcoma has got variable presentation due to variation in degree of cellularity , mitotic activity and pleomorphism which has led to the histological grading of the tumor .
grade i ( well differentiated ) refers to tumors of uniform nuclear appearance with an appreciable amount of collagenous intercellular substance . in grade ii ( intermediate )
grade iii ( high ) tumors are anaplastic cellular growths often containing many giant cells and mitotic figures .
histological grading is important , as it has direct correlation with the prognosis of tumor . occasionally , it is difficult to differentiate fibrosarcoma from other spindle - cell neoplasm .
markers for muscle ( desmin and actin ) , macrophages ( cd68 ) , neural tissue ( s-100 , neuron specific enolase ) , melanoma ( hmb-450 ) and epithelial tissue ( cytokeratin , epithelial membrane antigen ) will be absent .
wadhwan et al . in 2010 reported two cases of fibrosarcoma of maxilla which was diagnosed on the basis of herringbone pattern with positivity for vimentin .
dhanavelu et al . in 2012 reported a case in a 73-year - old female patient .
khanna et al . in 2014 reported a case of fibrosarcoma of maxilla with its extension into maxillary sinus .
the overall 5 year survival rate for fibrosarcoma of bone has been reported to be between 28.7% and 34% .
periosteal fibrosarcoma has a better prognosis than that of medullary origin , with 5 year survival rates of 52% and 27% , respectively .
local recurrence is very common , but metastasis is rare and occurs mostly in lungs or to distant bones .
fibrosarcoma of jaws are rare , detailed clinical , histological and immunohistochemical analysis is very important in such cases .
dentists should be able to recognize the features of fibrosarcoma to propose appropriate investigations and help in treatment planning .
| fibrosarcoma is a malignant tumor of fibroblasts . at one time , it was considered one of the most common soft tissue sarcomas .
however , the diagnosis of fibrosarcoma is made much less frequently today because of the recognition and separate classification of other spindle cell lesions that have similar microscopic features .
of all the fibrosarcomas occurring in humans , only 0.05% occur in the head and neck region . here , we present a case of 22-year - old female patient with the swelling on the left anterior aspect of the face .
histopathologically , the lesion was diagnosed as fibrosarcoma and immunohistochemically , the lesional cells showed positivity for vimentin . |
the intraband exciton dynamics of
molecular aggregates is a crucial
initial step to determine the possibly coherent nature of energy transfer
and its implications for the ensuing interband relaxation pathways
in strongly coupled excitonic systems . in this work ,
we fully characterize
the intraband dynamics in linear j - aggregates of porphyrins , good
model systems for multichromophoric assemblies in biological antenna
complexes . using different 2d electronic spectroscopy schemes together
with raman spectroscopy and theoretical modeling
, we provide a full
characterization of the inner structure of the main one - exciton band
of the porphyrin aggregates .
we find that the redistribution of population
within the band occurs with a characteristic time of 280 fs and dominates
the modulation of an electronic coherence .
while we do not find that
the coupling to vibrations significantly affects the dynamics of excitonic
coherence , our results suggest that exciton fluctuations are nevertheless
highly correlated . | |
diabetes mellitus is a common noncommunicable disease in india , as well as the rest of the world .
it has emerged as a major public health problem , with low- and middle - income countries facing the greatest burden . as of 2013
, india ranks second in the list of diabetes among people aged 2079 years next only to china .
india had 65.1 million diabetic people aged 2079 years , while china had 98.4 million people . probably because of a staggering rise in obesity , diabetes has manifested as a global epidemic . the change in life expectancy and lack of improvement in healthcare are in part responsible for the astounding rise in the incidence of this disease . even in the rural indian
, population is undergoing lifestyle transition due to socioeconomic growth which can also be cited as a reason for increasing incidence of diabetes in rural areas .
diabetes is a chronic disease , requiring a multipronged approach for its management , wherein the patient has an important role to play .
they are required to follow certain self - care practices to achieve an optimal glycemic control and prevent complications .
these practices include regular physical activity , appropriate dietary practices , daily foot care practice , compliance with treatment regimen , and tackling complications such as hypoglycemic episodes .
thus , the objective of this study was to assess the baseline knowledge and self - care behavioral practices regarding diabetes among the rural population so that it will serve as a benchmark for future comparisons to assess the effectiveness of any educational training program for the diabetic patients .
a community - based , cross - sectional study was done among patients with type 2 diabetes mellitus in sullia taluk , karnataka , to assess their knowledge regarding the disease and the self - care practices that were followed by them . assuming that 50% of the diabetics had reasonable knowledge , and they followed self - care practices as advised and requiring a precision of 5% , the sample size was calculated using the formula , 4 pq / d .
thus , the study was conducted among 400 patients with type 2 diabetes mellitus who were 20 years of age and above and who had the disease for at least 1 year and residing in that locality for more than a year .
the patients were selected from forty villages of sullia by probability proportionate to sample size and then by a random sampling technique .
a pretested , semi - structured questionnaire was handed over to each participant to collect sociodemographic details , diabetes - specific information , knowledge regarding diabetes , and the self - care practices that were followed by them .
the questionnaire was reviewed by three professionals before being used a public health expert , a diabetologist , and a statistician and their suggestions were utilized for improving the questionnaire thus ensuring consensus validity of the instrument .
the questionnaire originally made in english was translated and back translated to and from kannada to ensure appropriateness of translation .
ethical clearance was obtained from the institutional ethical committee before the actual start of the study .
knowledge was assessed using closed - ended questions , and the study subjects were classified as having poor , average , and good knowledge depending on the score obtained .
details regarding self - management activities were collected using the summary diabetes self - care activities questionnaire after making minor changes to it to suit the local study population .
the following variables were checked for : smoking habits , checking of feet daily and checking the inside of the footwear , checking blood sugars regularly and as advised by the health - care provider , regular drug intake , exercise for at least 5 days in a week , and adherence to a healthy eating plan . since self - care practices can not be scored collectively and given a single value , it was partitioned into different habits and then scored accordingly .
the data collected were entered in microsoft excel office 2007 and spss statistics version 20 ( ibm , new york , united states ) was used for statistical analysis .
majority of the respondents were males ( 61.2% ) and belonged to 4049 years age group ( 31% ) . majority of the study population belonged to hindu religion ( 71% ) and were married ( 91.2% ) .
most of the study population were either agriculturists or self - employed and belonged to the upper middle class as per modified bg prasad classification .
fifty - one percent of the respondents had diabetes for 15 years , 35% for 610 years , and 11% of them for 1120 years .
regarding the treatment profile of the study group , it was evident that 50% were treated by doctors with mbbs qualification and < 1% by specialists .
characteristics of the study population among the participants , almost half of them ( 49.5% ) denied of having any comorbidity . among those who knew of comorbidities in them , majority of them accepted to have hypertension ( 32% ) followed by dyslipidemia ( 28.75% ) .
the study population was questioned about the general features of diabetes , its risk factors , symptoms , mode of diagnosis , and complications to estimate the knowledge about the disease .
it was seen that only 24% of the participants had good knowledge about the disease , 59% of them had an average knowledge , and 17% had poor knowledge . among the general questions that were asked on diabetes , more than 50% had good knowledge on diabetes being hereditary ( 65.25% ) and
more than 50% of the study population did not know that age , high cholesterol , and low physical activity were risk factors of diabetes .
about 50% of them did not know that fatigue , hunger , and thirst could be a symptom of diabetes . more than 70% of them were not aware that neuropathy , skin infection , and ophthalmic problems could be a complication of diabetes [ table 2 ] .
frequency distribution of respondent 's knowledge regarding diabetes mellitus more than 50% were aware that fatigue ( 77% ) , excessive sweating ( 67.5% ) , and blurred vision ( 55.75% ) were signs of hypoglycemia .
however , < 50% of them were aware that a headache ( 35.25% ) could be a symptom of hypoglycemia [ table 2 ] .
more than 80% felt that diabetes can be detected by blood sugar estimation [ table 2 ] .
regarding misconceptions about diabetes , more than 50% of the respondents ( 56.5% ) felt that drugs can be stopped once diabetes is controlled .
it was noted that those participants who visited private establishments for their treatment were more knowledgeable than those who visited government facilities , and this was also found to be statistically significant ( = 9.09 , df = 1 , p = 0.003 ) .
participants who visited health - care providers of modern medicine background ( allopathic practice ) were found to be more knowledgeable than those who visited ayush practitioners , and this was also found to be statistically significant ( = 12 , df = 1 , p = 0.001 ) .
anova was done to compare the mean knowledge score obtained against various age groups , religion , educational status , occupation , and socioeconomic status .
z - test was done to compare the knowledge score obtained between the two genders .
it was noted that there was statistically significant difference in the knowledge among age groups ( f = 4.041 , p < 0.05 ) , religion ( f = 34.67 , p < 0.05 ) , occupation ( f = 17.54 , p < 0.05 ) , socioeconomic status ( f = 9.41 , p < 0.05 ) , and duration of diabetes ( f = 7.33 , p < 0.05 ) . among the self - care practices ,
it was seen that checking the feet daily and inspecting the inside of shoes / footwear daily were the two practices which were not followed by a majority of the study participants which accounted for 99.5% .
the practices that were best followed were checking of blood sugars at least once in 3 months and as advised by the doctor which accounted for almost 65% and 73% of the respondents .
only 48% of the respondents were regular in taking drugs and 20.5% exercised for at least 5 days in a week for 2030 min .
the details of the adherence to self - care practices are given in table 3 .
self - care practices among respondents logistic regression analysis of each self - care practice with blood sugars was done , and the results are shown in table 4 .
it was seen that dietary practice is one of the important factors influencing the achievement of good blood sugars in the participants .
the present study was done to assess the knowledge and practices regarding diabetes among the known diabetics residing in rural field practice area of a teaching hospital .
most of the study participants were in the 4049 years age group , which was slightly lower than that seen in studies done by priyanka and angadi and shah et al .
the age group is higher when compared to studies conducted in two places of karnataka , namely kolar and dharwad , by muninarayana et al . and patil et al .
, respectively , where the majority of the participants were in the age group of 3045 and 3049 years , respectively . in the present study ,
the duration of diabetes in the participants was mostly 15 years in contrast to study done by hawal et al . where the majority of them had diabetes for more than 5 years .
regarding the literacy status of the study participants , it was found that only 9.5% were illiterates as compared to 36.64% as seen in the study done by shah et al .
it is generally thought that the duration of diabetes and a literacy rate of the participants have some influence on the knowledge regarding the disease . in our study ,
higher the education of the participant better was the knowledge on the disease but lesser the duration of the disease , higher the knowledge .
one of the most important findings in this study is that only 24% of the respondents had an overall good knowledge regarding diabetes .
this is a matter of concern because india has around 65.1 million diabetic people and poor knowledge about their own health status and disease may be one of the barriers for healthful living
. further knowledge can serve as an important resource base for improving their own health and that of the society .
the results of our study regarding knowledge of the participants are similar to chennai urban rural epidemiology study ( cures-9 ) study which also reflects the poor knowledge and awareness about diabetes among the chennai population .
there was a misconception among 72% of our study participants that all bitter substances can treat diabetes against 53% which was seen in a study done by shah et al . in saurashtra .
eighty - two percent knew the methods to diagnose diabetes mellitus which was slightly different from findings in a study done by gupta et al .
where 90% of the study population in the rural area knew about accurate methods to diagnose diabetes .
again , knowledge regarding complications of diabetes was also poor , which was also similar to cures study .
only 30% of the participants knew that diabetes can cause ophthalmic problems against 15% of the participants in cures study .
the results obtained in our study regarding the knowledge on complications were different from the results obtained by mehta et al . , who observed that 82% of his study subjects had knowledge about the disease and its complications .
it was observed that mean composite knowledge score was better in the 3049 years age group , being a professional and belonging to upper class .
with respect to the age group who also found that younger the age group , better the knowledge .
it was also seen that those with < 5 years of disease had a better knowledge score .
this is probably because the younger generation is more educated , aware , and more exposed to the media which make them knowledgeable .
this poor knowledge indicates that most of them are not educated regarding their disease by their primary care physicians and field - level health workers .
one of the reasons for the lack of providing education could be that field workers are themselves not aware or not motivated to educate the public .
physician 's failure in this part may be due to the heavy load of patients that they see in their daily practice and thus the lack of time to educate .
this emphasizes that the already existing field - level workers like ashas should be educated , trained , and motivated in this aspect .
education through mass media can also bring about a change . among the self - care practices , good dietary behavior was present only in 24% of the study participants .
this was quite different from a study done by rajasekharan et al . in an urban area where 46% of the participants followed a diet plan regularly .
emphasis should be given to good dietary practices as it is a determinant of both glycemic control and weight management .
the world health organization recommends at least 400 g of fruits and vegetables per day which was not followed by most of the study participants .
consumption of these minimum recommended levels of fruits and vegetables will also protect an individual from cardiovascular diseases , stroke , and gastrointestinal diseases . in our study , only 3% of the participants consumed the recommended fruits and vegetables for at least 5 days or more in a week .
where 26% of the participants included fruits and vegetables in their diet on all days of the week .
however , it was interesting to see that only 4% of the study participants included high - fat diet on 5 or more days of the week .
these findings are almost similar to those seen in studies done by rajasekharan et al . and gopichandran et al .
regarding the physical activity , only 19% of the study participants followed the recommended 2030 min exercise per day for at least 5 days a week .
regularly will have many benefits ranging from reduced insulin resistance , blood pressure control , and cardioprotective role .
sixty - five percent of the study participants checked their blood sugars at least once in 3 months .
emphasis should be laid on checking blood sugars as the effectiveness of the treatment regimen can be ascertained only by checking their blood sugars .
forty - eight percent of the participants take the recommended hypoglycemic agents and insulin daily and regularly , which were lower than the study conducted elsewhere .
regarding foot care , only 0.5% of them checked their feet and inspected the inside of their shoes daily which is < 9% and 12% , respectively , which was observed in a study done by raithatha et al .
none of them wore footwear inside their houses , which was in contrast to a study done in mumbai by chandalia et al .
logistic regression of self - care practices against the blood sugar levels showed that dietary practice is one of the important factors influencing the maintenance of good blood sugar levels in the participants .
these findings are slightly different from a study conducted by wynn nyunt et al . where following a healthy diet , following exercise for at least 5 days in a week , and compliant to drug therapy were all associated statistically with the achievement of glycemic control .
as evidenced by the study , it was noted that only about one - fourth of diabetics had a good knowledge .
the respondents were very poor with regards to the daily checking of foot and inside of footwear and also in adherence to exercises .
although the indian urban population has access to reliable screening methods , antidiabetic medications , counseling services , and preventive services , such health benefits , are not often available to the rural patients .
there is a disproportionate allocation of health resources between urban and rural areas and in addition , poverty in rural areas may be responsible for this poor knowledge and self - care practices .
food insecurity , illiteracy , poor sanitation , and dominance of communicable diseases may also contribute which suggests that both policy makers and local governments may be undermining and underprioritizing the looming threat of diabetes .
such inadequacies contribute to an infrastructure that may result in poor diabetes screening and preventive services , nonadherence to diabetic management guidelines , lack of available counseling , and long distance travel to health services .
regular health education services which will make them knowledgeable about the disease and encouraging self - care management in those who are diagnosed as diabetic will reduce the health - care burden and help in achieving glycemic control and thus minimizing complications . as this study
is done to assess the knowledge and self - care practices among diabetic patients only , the results can not be generalized to the whole community .
one more limitation is that the blood sugar values that have been obtained for this study are not done by the author .
the last blood sugar values done by the patient at different times have been taken for this study . and also
the questionnaire that is used is closed ended , and they can sometimes be guessed by the respondents .
as this study is done to assess the knowledge and self - care practices among diabetic patients only , the results can not be generalized to the whole community .
one more limitation is that the blood sugar values that have been obtained for this study are not done by the author .
the last blood sugar values done by the patient at different times have been taken for this study . and also the questionnaire that is used is closed ended , and they can sometimes be guessed by the respondents .
| introduction : diabetes is a lifestyle disease which requires a multipronged approach for its management , wherein patient has an important role to play in terms of self - care practices , which can be taught to them by educational programs . to develop such an educational program , a baseline assessment of knowledge and self - care practices of patients , needs to be made .
the two objectives of the study were to estimate the knowledge of diabetic patients regarding the disease and its complications , and to estimate the knowledge and adherence to self - care practices concerned with type 2 diabetes mellitus.methods:the study was conducted in rural sullia , karnataka , from january 2014 to may 2015 .
the sample size was calculated to be 400 , and the sampling method was probability proportionate to sampling size.result:majority of them were married males of hindu religion and belonged to upper middle class .
only 24.25% of them had good knowledge . among the self - care practices , foot care was the most neglected area.conclusion:only one - fourth of the study population had a good knowledge toward diabetes .
adherence to some of the self - care practices was also poor .
government policies may help in creating guidelines on diabetes management , funding community programs for public awareness , availability of medicines , and diagnostic services to all sections of the community .
continuing education programs for health - care providers and utilization of mass media to the fullest potential may also help in creating awareness . |
key clinical messagediagnosing hsp can be difficult , especially when abdominal symptoms precede the onset of characteristic palpable purpura ( chen mj et al .
2005 , world gastroenterol . , 11 , 2354 )
.
therefore , it is necessary to consider the possibility of hsp in patients with prolonged strong abdominal pain , even in cases without purpura . | |
regulated upon activation normal t - cell expressed and secreted ( rantes ) is a key signaling cytokine involved in initiation of physiologic inflammation ( conti et al . , 1999 ) as well as in many pathologic inflammatory conditions including endometriosis ( oral et al . , 1996 ) .
rantes has been shown to be responsible for 70% of the stimulation of monocyte migration caused by peritoneal fluid from women with endometriosis ( hornung et al . , 2001a ) .
some authors have suggested that rantes production might be inhibited by activation of the ppar pathway .
thiazolidinediones including rosiglitazone and pioglitazone are a new class of insulin sensitizers that do not stimulate insulin secretion ( adams et al . , 1997 ) .
these drugs act as ppar- agonists , alter gene expression , enhance the sensitivity of tissue to insulin , and partially reverse lipid abnormalities in patients with diabetes ( lenhard , 2001 ) .
therefore , theses drugs have been used for patients with polycystic ovary syndrome ( pcos ) undergoing ivf treatment .
our previous study demonstrated that pioglitazone therapy reduces intraovarian stromal blood flow and is beneficial in improving both the response to ovarian stimulation and ivf outcome in pcos patients undergoing ivf ( kim et al . , 2010 ) .
it has been reported that ppar- also has a beneficial effect on endometriosis . in an in vitro study
, ppar- reduced the monocyte migration induced by peritoneal fluid from women with endometriosis ( hornung et al . , 2001b ) .
therefore , we performed this study to investigate the effect of pioglitazone , a drug of the thiazolidinedione class , on production of rantes and in vitro fertilization - embryo transfer ( ivf - et ) outcomes in infertile patients with stage ii or iv endometriosis undergoing ivf treatment .
sixty - four infertile patients with stage iii or iv endometriosis who underwent ivf were prospectively recruited to this randomized study , which was performed at the university - based infertility clinic of the asan medical center , seoul , south korea .
all subjects were diagnosed by laparoscopic surgery and histologic examination with stage iii or iv endometriosis according to the revised american society for reproductive medicine ( asrm ) classification .
patients were randomly allocated to the treatment group ( n=32 ) or the placebo group ( n=32 ) by number allocation using sealed envelopes .
all patients had endometriosis , and had failed to ovulate or conceive after repeated treatment with clomiphene citrate or gonadotropin .
all patients were in good health with normal thyroid , hepatic , and renal function .
blood samples were collected from each patient on menstrual cycle day ( mcd ) 3 for basal measurement of sex hormone and prolactin levels .
the long protocol of gnrh agonist ( gnrh - a ) was used for cos in all patients .
daily injection of a gnrh agonist 1 mg ( leuprorelin acetate , lorelin ; dongkook , korea ) was initiated commencing at the midluteal phase and was continued until menses , followed by a dose reduction to 0.5 mg daily .
ovarian stimulation was started with 50150 iu of recombinant human follicle - stimulating hormone ( rhfsh , puregon ; organon , oss , the netherlands ) on menstrual day 3 .
the initial dose of rhfsh was calculated with reference to patient age and body mass index .
when one or more follicles attained a mean diameter of 18 mm , 250 g hcg was subcutaneously administered to induce follicular maturation . in the pioglitazone treatment group , pioglitazone ( actos ; takeda , osaka , japan ) , 15 mg once daily ,
was given on the commencement day of gnrh - a administration to the day of human chorionic gonadotropin ( hcg , ovidrel ; serono , geneva , switzerland ) injection .
oocytes were retrieved 3536 hours later , and one to four embryos were transferred to the uterus three days later . during the luteal phase , 90 mg of vaginal progesterone gel ( crinone 8% ; serono ) was applied once daily , starting on the day of oocyte retrieval .
pregnancies were identified by an increase in serum -hcg concentration and transvaginal ultrasonographic evidence of a gestational sac .
serum concentrations of -hcg were measured 11 days after embryo transfer ( et ) by radioimmunoassay using an hcg maia clone kit ( serono diagnostics , woking , uk ) ; the inter- and intra - assay variances values were < 10% and 5% , respectively .
blood samples were drawn for serologic assay of rantes on the day on which the gnrh agonist was first administered and the day of hcg injection .
rantes was measured using a solid - phase enzyme - linked immunosorbent assay ( medgenix diagnostics , fleurus , belgium ) ; interassay and intraassay variances were less than 10% and 5% , respectively .
the chi - squared test , fisher s exact test , the mann - whitney u test and spearman s coefficient of correlation were used , as appropriate , in comparisons .
statistical significance was defined as p<0.05 . the spss statistical package for windows , version 11.0 ( spss inc , chicago , il ) was used for all analyses .
sixty - four infertile patients with stage iii or iv endometriosis who underwent ivf were prospectively recruited to this randomized study , which was performed at the university - based infertility clinic of the asan medical center , seoul , south korea .
all subjects were diagnosed by laparoscopic surgery and histologic examination with stage iii or iv endometriosis according to the revised american society for reproductive medicine ( asrm ) classification .
patients were randomly allocated to the treatment group ( n=32 ) or the placebo group ( n=32 ) by number allocation using sealed envelopes .
all patients had endometriosis , and had failed to ovulate or conceive after repeated treatment with clomiphene citrate or gonadotropin .
all patients were in good health with normal thyroid , hepatic , and renal function .
blood samples were collected from each patient on menstrual cycle day ( mcd ) 3 for basal measurement of sex hormone and prolactin levels .
the long protocol of gnrh agonist ( gnrh - a ) was used for cos in all patients . daily injection of a gnrh agonist 1 mg ( leuprorelin acetate , lorelin ; dongkook , korea ) was initiated commencing at the midluteal phase and was continued until menses , followed by a dose reduction to 0.5 mg daily .
ovarian stimulation was started with 50150 iu of recombinant human follicle - stimulating hormone ( rhfsh , puregon ; organon , oss , the netherlands ) on menstrual day 3 .
the initial dose of rhfsh was calculated with reference to patient age and body mass index .
when one or more follicles attained a mean diameter of 18 mm , 250 g hcg was subcutaneously administered to induce follicular maturation . in the pioglitazone treatment group , pioglitazone ( actos ; takeda , osaka , japan ) , 15 mg once daily ,
was given on the commencement day of gnrh - a administration to the day of human chorionic gonadotropin ( hcg , ovidrel ; serono , geneva , switzerland ) injection .
oocytes were retrieved 3536 hours later , and one to four embryos were transferred to the uterus three days later . during the luteal phase , 90 mg of vaginal progesterone gel ( crinone 8% ; serono ) was applied once daily , starting on the day of oocyte retrieval .
pregnancies were identified by an increase in serum -hcg concentration and transvaginal ultrasonographic evidence of a gestational sac .
serum concentrations of -hcg were measured 11 days after embryo transfer ( et ) by radioimmunoassay using an hcg maia clone kit ( serono diagnostics , woking , uk ) ; the inter- and intra - assay variances values were < 10% and 5% , respectively .
blood samples were drawn for serologic assay of rantes on the day on which the gnrh agonist was first administered and the day of hcg injection .
rantes was measured using a solid - phase enzyme - linked immunosorbent assay ( medgenix diagnostics , fleurus , belgium ) ; interassay and intraassay variances were less than 10% and 5% , respectively .
the chi - squared test , fisher s exact test , the mann - whitney u test and spearman s coefficient of correlation were used , as appropriate , in comparisons .
the spss statistical package for windows , version 11.0 ( spss inc , chicago , il ) was used for all analyses .
patients clinical characteristics and baseline hormone concentrations were similar in the pioglitazone and control groups ( table 1 ) .
there were no differences between the two groups in the total dose of rhfsh administered , and the numbers of oocytes retrieved , oocytes fertilized and grade i or ii embryos .
the clinical pregnancy rate was somewhat higher in the pioglitazone group of 37.5% compared with 31.3% of the control group , but the difference did not achieve the statistical significance . however , the embryo implantation rate was significantly higher in the pioglitazone group than in the control group ( 13.8% vs. 8.6% , p<0.01 ) .
patient characteristics comparison of controlled ovarian stimulation results and ivf - et outcome when cos results and ivf outcomes were compared between pregnant and non - pregnant subgroups of the pioglitazone group , there were no significant differences between pregnant and non - pregnant subgroups in the numbers of oocytes retrieved , mature oocytes , oocytes fertilized and embryos transferred .
rantes concentrations after pioglitazone treatment decreased significantly compared with that before pioglitazone treatment ( p<0.01 ) . however , rantes concentration before and after placebo treatment was not different in the control group . in the pregnant subgroup of the pioglitazone group ,
rantes concentration before pioglitazone treatment was significantly lower than in the non - pregnant subgroup ( p<0.05 ) .
after pioglitazone treatment , rantes concentrations decreased significantly in both subgroups ( p<0.01 in the pregnant subgroup , p<0.05 in the non - pregnant subgroup ) .
plasma rantes levels in patients with endometriosis who underwent ivf - et before and after pioglitazone treatment .
endometriosis is an inflammatory disease characterized by the presence of endometrial glandular and stromal cells in areas outside of the uterine corpus .
the reported disease prevalence varies widely depending on the population studied and the method of sampling employed , the prevalence of endometriosis appears to range from 2% to 18% , whereas within infertile populations the prevalence has been reported to be as high as 50% ( missmer & cramer , 2003 ) .
endometriosis is associated with activation of immune cells and aberrant cytokine production in peritoneal fluid , creating a local inflammatory environment ( lebovic et al . , 2001 ;
in pathologic states including chronic endometritis and endometriosis , inflammation followed by cellular infiltration into the area of interest and is followed by differentiation and activation of immune cells ( pritts et al . , 2002 ) .
such cells secrete growth factors , cytokines , prostanoids , complement components , and hydrolytic enzymes ( oral et al . , 1996 ) .
the levels of cytokines associated with inflammation have been reported to be higher in women with endometriosis ( bedaiwy et al . ,
2002 ) as well as in those with unexplained infertility ( demir et al . , 2009 ) .
both ectopic and eutopic endometrium can be stimulated to produce materials chemoattracting mononuclear cells , including monocyte chemotactic protein-1 ( mcp-1 ) ( arici et al . , 1997 ) ,
interleukin-8 ( il-8 ) ( arici et al . , 1996 ) and rantes ( khorram et al . , 1993 ) .
many studies have demonstrated abnormal local endometrial production of pro - inflammatory cytokines , such as tumor necrosis factor a ( tnf - a ) , il-1 , il-6 , and il-8 ( koninckx et al . , 1999 ) .
increasing evidence indicates that rantes is the key chemokine involved at early stages of the inflammatory process ( appay & rowland - jones , 2001 ) .
one report showed that rantes is responsible for 70% of the in vitro monocyte migration activity in peritoneal fluid from women with endometriosis ( hornung et al . , 2001 ) .
subsequent activation of cellular inflammatory mediators might be prevented by blocking of early monocyte infiltration .
ppar plays a key role in the regulation of inflammation and insulin secretion ( yessoufou et al . , 2006 ) .
( 2001 ) demonstrated transcription of ppar- in rat granulosa cells . as well as exerting multiple effects on insulin production ,
recently thiazolinediones have been shown to exhibit both insulin - independent and insulin - sensitizing direct effects on steroidogenesis and production of igf binding protein-1 in human ovarian cells .
this implies that ppar- is expressed in the human ovary ( seto - young et al . , 2005 ) .
it has been reported that ppar- also affect rantes secretion even in the endometrial stromal cells ( pritts et al . , 2002 ) .
the cited in vitro study suggested that ppar inhibits the pathway leading to rantes expression in endometrial stromal cells . by binding to rosiglitazone
, ppar- receptor - ligand complexes seem to interact with response elements on the rantes gene promoter , thereby blocking the subsequent transcription and translation of rantes ( pritts et al . , 2002 ) .
however , studies on the effect of ppar- or ppar- agonist on rantes secretion in endometriosis or endometrial tissue are very limited . moreover , it is hard to find out the study on the effect of ppar- agonist on rantes and ivf outcome in patients with endometriosis .
our study is the first report on the effect of thiazolinediones on rantes secretion and ivf outcome in infertile women with advanced endometriosis undergoing ivf treatment . in the present study
, we used a pioglitazone as a ppar- ligand to treat infertile patients with endometriosis and evaluated whether ppar- ligand such as pioglitazone could inhibit production of rantes via activation of the ppar pathway and improve ivf outcomes in infertile patients with advanced endometriosis undergoing ivf .
as we expected before study , pioglitazone had a beneficial effect on rantes secretion and ivf outcome in advanced enometriosis patients .
the thiazolidinediones including rosiglitazone and pioglitazone belong to a new class of insulin sensitizers that do not stimulate insulin secretion .
the first available drug of the thiazolidinedione class , troglitazone , was effective to induce ovulation in patients with polycystic ovary syndrome ( pcos ) , but was later reported to be associated with fatal hepatotoxicity ( azziz et al . , 2001 ) .
rosiglitazone , another thiazolidinedione , has also been shown to be effective in inducing ovulation in pcos patients .
however , the fda encourages healthcare professionals to follow current recommendations on the drug label , including a contraindication of rosiglitazone use in patients with nyha class iii or iv heart failure , and avoidance of use in patients with symptomatic heart failure .
all patients should be monitored for signs and symptoms of heart failure ( rapid weight gain , difficulty in breathing , and/or swelling ) after initiation of treatment and after an increase in dose ( lago et al .
although the response to ovarian stimulation did not improve , the implantation rate was significantly higher in the treatment group .
further , in the pregnant subgroup , rantes concentration was significantly lower than in the non - pregnant subgroup .
statistically significant differences in rantes concentrations were evident before and after treatment . however , however , our study has a limitation to evaluate the efficacy of pioglitazone treatment in endometriosis patients due to a small number of sample available .
therefore , well - designed larger studies with longer follow - up times are needed to confirm the effectiveness and safety of pioglitazone . | this study was performed to investigate the effect of peroxisome proliferators activated receptor- ( ppar- ) ligand , pioglitazone , on production of regulated upon activation normal t - cell expressed and secreted ( rantes ) and in vitro fertilization ( ivf ) outcome in infertile patients with endometriosis .
sixty - four infertile patients with stage iii or iv endometriosis undergoing ivf were randomly allocated to the study or the control group .
the long protocol of gnrh agonist ( gnrh - a ) was used for controlled ovarian stimulation ( cos ) in all patients .
patients in the study group were treated with pioglitazone at a dose of 15 mg / day orally from the starting day of gnrh - a treatment to the day of hcg injection .
blood samples were drawn for serologic assay of rantes on the first day of gnrh - a treatment and the day of hcg injection .
there were no differences between the study and control groups in patient characteristics .
there were also no differences between the two groups in cos duration , and the numbers of retrieved oocytes , fertilized oocytes and embryos transferred .
the clinical pregnancy rate per cycle was higher in the study group , but this difference was not statistically significant . however , embryo implantation rate was significantly higher in the study group of 12.5% compared with 8.6% in the control group ( p<0.05 ) .
the serum rantes levels after pioglitazone treatment were significantly lower than those before pioglitazone treatmen in the study group ( p<0.05 ) .
our data suggest that pioglitazone treatment can suppress rantes production and improve the embryo implantation rate in patients with endometriosis undergoing ivf . |
the deletion of long arm of chromosome 5 ( del(5)(q32 ) ) is a very rare event in acute lymphoblastic leukemia ( all ) and has not been analyzed extensively .
the del ( 5)(q ) is associated with 20 - 30% of mds , either as the sole identifiable abnormality , or in combination with one or more additional abnormalities.[14 ] although , this particular abnormality is common to myeloid lineage associated neoplasias , nevertheless , we present a case of t - all with del(5)(q32 ) as a sole chromosomal abnormality with case history and discussed with relevant literature .
a 75-year - old male patient presented to the opd of bangalore institute of oncology with a one month history of intermittent fever and loss of appetite .
there was no history of bleeding tendency , diabetes mellitus , hypertension and ihd . on physical examination , his abdomen ,
complete blood count showed hb12.5g / dl , wbc 130.46 10/l , neutrophils 2% , lymphocytes 20% , blasts 78% , rbc 4.33 10/l , platelet count 127 10/l and ldh was high with an activity of 1949u / l .
immunophenotyping performed on bone marrow aspirate with a gating on ssc / fsc showed majority of the gated cells to be strongly expressing cd45 , cd5 , cd10 , cd7 , ccd3 , cd34 , and cd13 and weakly positive or negative for cd14 , cd33 , cd19 , cd3 , hla - dr , cd117 , cd8 , cd22 , cd4 , ccd22 , mpo and tdt . the scattered parameters and antigen profile as analyzed by flow cytometry ( cyan - dako ) correlated with morphology and diagnosis of t - acute lymphoblastic leukemia ( calla + ve )
the bone marrow aspirate was cultured for direct and 24hrs in rpmi-1640 medium supplemented with 20% qualified ; heat inactivated fetal bovine serum , 100 u / ml penicillin and streptomycin , without any mitogen at 37c .
the cultures were exposed to colcemid ( final concentration- 0.1 g / ml ) for 30 minutes followed by hypotonic treatment ( 0.075 m kcl ) for 20 minutes at 37c and fixed in methanol : glacial acetic acid ( 3:1 ) overnight at 4c . later , air dry slides were made and incubated at 60c overnight for aging .
a total of 25 metaphases were screened , captured , karyotyped and analyzed using applied spectral imaging software ( asi ) .
all 25 metaphases consistently showed a karyotype of 46 , xy , del(5)(q32 ) [ figure 1 ] .
del(5)(q32 ) is a most frequent and documented recurrent chromosome abnormality with favourable prognosis in mds.[13468 ] this is also reported in acute myeloid leukemia ( aml ) transformed from mds ( 1 ) .
the rarity of ( 5)(q ) deletion in all and the same abnormality in our patient persuaded us to explore the literature .
1992 ) have reported three cases : del(5)(q ) in all with biphenotypic and early progenitor phenotype as sole abnormality in the first case , as an evolutionary event in another and with ph positivity in a third case .
in contrast to its presence in aml , del(5 ) ( q ) in all is not an adverse prognostic indicator , and it appears to be more frequent in children .
our patient is an adult and has been diagnosed as having t - all by flow cytometry and all - l2 by morphology .
the patient is undergoing treatment and will be followed up to evaluate the prognostic significance of del(5)(q ) .
literature review reveals , that del(5)(q ) is also reported in chronic lymphocytic leukemia ( cll ) .
however , they are rare and been reported only as karyotypic results without known prognosis .
( 2010 ) describe two cll cases with del(5)(q ) not associated with adverse prognosis and not related to induced chromosome changes .
this abnormality is not only reported in leukemia but also reported in small cell neuroendocrine lung carcinoma .
these are difficult to estimate by conventional cytogenetic analysis , because small differences in base pairs at deletions are beyond the sensitivity of the technique .
further studies are required to elucidate the prognostic value of del(5)(q ) in more all patients and to identify candidate genes that may play a vital role in the pathogenesis of all . | del(5)(q ) is a common chromosomal abnormality with favourable prognosis in myelodysplastic syndrome ( mds ) and acute myeloid leukemia ( aml ) .
however , del(5)(q ) is also seen rarely in acute lymphoblastic leukemia ( all ) and its significance remains poorly understood .
we present here , a case report of diagnosis of an adult 75 year old patient of all with a cytogenetic abnormality of del(5)(q32 ) .
his clinical features , morphology and immunophenotyping findings were suggestive of t - all .
relevant literature has been reviewed and discussed . |
because of the improved culture methods ( 8,9 ) and sensitive nucleic acid amplification assays ( naaas ) ( 1014 ) developed in recent years , kingella kingae , a gram - negative coccobacillus of the neisseriaceae family , is increasingly recognized as an invasive pathogen of early childhood .
the organism is a frequent source of childhood bacteremia and the most common agent of skeletal system infections in children 6 months3 years of age ; it is also a cause of bacterial endocarditis in children and adults ( 816 ) .
because of the fastidious nature of k. kingae , many illnesses caused by this bacterium are , probably , overlooked .
although most cases of invasive k. kingae infections are sporadic , clusters of invasive disease have been detected among attendees of daycare centers in israel , europe , and the united states .
the bacteriologic identification of k. kingae relies on the following : typical gram stain results , showing pairs or short chains of plump , gram - negative bacilli with tapered ends ; -hemolysis ; pitting of the agar surface ; failure to grow on macconkey medium ; weak oxidase activity and a negative catalase reaction ; production of acid from glucose ; and , with rare exceptions , production of acid maltose ( 8,9 ) .
however , k. kingae tends to retain crystal violet dye and , therefore , it may appear to be gram - positive , and laboratories unfamiliar with its cultural and staining features may misidentify the bacterium altogether or dismiss invasive isolates as culture contaminants .
identification of k. kingae , however , is not difficult , and many commercial instruments and technologies such as vitek 2 ( biomrieux , marcy - letoile , france ) , matrix - assisted laser desorption ionization - time of flight mass spectrometry ( maldi - tof ) , or 16s rdna gene sequencing , correctly identify the organism .
recent studies have demonstrated the presence of a polysaccharide capsule on the surface of k. kingae that exhibits between - strains chemical variation and , most probably , antigenic variation ( 17,18 ) .
these characteristics may assist k. kingae in evading the immune response by facilitating the successive colonization of the host by strains harboring different capsular types .
the polysaccharide capsule probably enables mucosal colonization , the survival of the organism in the bloodstream , and invasion of deep body sites ( 17,18 ) .
in addition , all k. kingae strains produce and secrete a potent repeats - in - toxin ( rtx ) that exhibits a wide range of cytotoxic activity and is particularly deleterious to macrophage - like cells , leukocytes , and synovial cells and , to a lesser degree , to respiratory epithelial cells that improve the organism s chances of surviving in the host and of invading skeletal tissues ( 12,19 ) .
similar to meningococci , k. kingae is carried on the oropharyngeal epithelium ( 20,21 ) , and the colonized mucosa is the portal of entry of the organism to the bloodstream from which it may disseminate to 3 areas for which the bacterium shows particular tropism : joints , bones , or the endocardium ( 22,23 ) .
damage to the upper respiratory surfaces by previous or concurrent viral infections or stomatitis appears to facilitate bloodstream invasion by k. kingae ( 16 ) .
k. kingae isolates show remarkable genomic diversity and , to date , 37 multilocus sequence typing ( mlst ) and 74 pulsed field gel electrophoresis ( pfge ) clones have been identified ( 2426 ) .
carried k. kingae organisms differ in their invasive capabilities ( 27 ) , and simultaneous carriage of 1 genotype is unusual ( 28 ) . whereas some strains , which are frequently isolated from healthy carriers , are seldom if ever detected in patients with clinical disease , others are rarely carried asymptomatically , but are responsible for a high proportion of invasive disease ( 24,27,28 ) .
however , a few strains appear to possess an optimal balance between transmissibility and invasiveness and are common among healthy carriers and among infected patients ( 24,27,28 ) .
a recent study has found that certain virulent k. kingae clones , characterized by a distinct combination of pfge and mlst profiles , are substantially associated with bacteremia with no focal infection , skeletal system invasion , or endocarditis , which suggests biological specialization for invading specific host tissues ( 25 ) .
most young children in whom an invasive k. kingae disease developed have been otherwise healthy . in contrast
, children > 4 years of age and adults who become infected frequently have underlying conditions such as congenital heart diseases , chronic renal failure , or a variety of primary immunodeficiencies ( 16 ) .
the prevalence rate in healthy children during the second year of life ranges between 10% and 12% ( 7,28 ) , which coincides with the peak attack rate of invasive infections ( 16 ) .
pharyngeal carriage of k. kingae and occurrence of disease before a child is 6 months of age are exceptions , indicating that maternal immunity and limited social contact provide protection ( 7,16,30 ) .
the clinical features of invasive k. kingae infections ( other than endocarditis ) are usually mild , and diagnosis requires a high level of suspicion by clinicians .
many patients with k. kingae associated joint or bone disease are afebrile and blood leukocyte counts , c - reactive protein levels , and erythrocyte sedimentation rates are frequently normal ( 16,31,32 ) .
although bacteremia with no focus is the second most frequent manifestation of invasive k. kingae infections , the condition is probably unsuspected and the diagnosis is likely missed in a large number of cases .
the current guidelines for managing illness in young , febrile children with no apparent source of infection , which use body temperature and leukocyte count as criteria for obtaining blood cultures ( 33 ) , are not sensitive enough for detecting occult k. kingae bacteremia because many infected children have a low - grade fever and may or may not have leukocytosis .
detection of k. kingae infections by culture is highly dependent on the use of adequate laboratory techniques .
the recovery of k. kingae from synovial fluid and bone exudates seeded onto routine solid culture media is suboptimal ( 8) . the yield of cultures can be substantially improved by inoculating clinical specimens into aerobic blood culture vials ( bcvs ) from a variety of commercial systems ( 8) .
attempts to isolate the organism from synovial fluid or bone exudates on routine solid media succeeded in 2 of 25 patients , whereas inoculation of these specimens into aerobic bactec ( becton dickinson , cockeysville , md , usa ) bcvs yielded the organism in all cases after a median incubation of 4 days ( 8) .
when specimens from bcvs determined to be positive by the automated blood culture instrument were subcultured onto a blood agar plate of trypticase soy agar with 5% sheep blood hemoglobin or chocolate agar , k. kingae grew readily , indicating that routine solid media are able to support the nutritional requirements of the organism .
this observation suggests that skeletal system exudates exert a detrimental effect on this fastidious bacterium , and dilution of purulent material in a large volume of broth decreases the concentration of inhibitory factors , improving its recovery ( 8) . in studies conducted in israel and france in which bcvs were routinely inoculated with synovial fluid aspirates from young children who had arthritis , k. kingae was isolated in 48% of the patients with culture - proven disease ( 8,9 ) .
conversely , when bcvs are not used , many k. kingae infections will be overlooked and labeled as culture - negative septic arthritis ( 34 ) . in recent years ,
development of naaas has further improved the diagnosis of k. kingae from skeletal system exudates .
use of this novel technique facilitates detection of difficult - to - culture organisms , enables bacteriologic diagnosis in patients already treated with antibacterial drugs , reduces time to detection , and facilitates precise identification of unusual species ( 1014 ) .
the procedure consists of extraction of bacterial dna from the synovial fluid sample , a pcr amplification step in which primers target the 16s rdna , the 23s rdna , or the rpo genes that are ubiquitous to all bacteria , then sequencing of the amplicon , and comparison of results with those kept in a broad database curator ( such as genbank ) to enable precise species identification .
alternately , the specimen may be subjected to amplification by pcr by using species - specific primers that recognize the most plausible pathogens .
use of naaas has confirmed that in countries where this topic has been studied , k. kingae is the most common etiologic agent of septic arthritis in children <3 years of age .
specific dna sequences , including in cases in which seeding of synovial fluid specimens into bcvs failed to recover the organism and shortened the time required to detect and identify the bacterium from 34 days to < 24 hours ( 1014 ) .
it was , then , natural that this sensitive approach was consequently adopted to study respiratory colonization by k. kingae and its connection to invasive disease . in an outbreak investigated by bidet et al .
, the cause of the skeletal infections was determined by sequential use of real - time pcr targeting the k. kingae
the same method was used to investigate the prevalence of k. kingae among attendees of the index daycare facility .
the organism was recovered by culture in 6 asymptomatic carriers , whereas the naas detected 5 additional carriers ( 35 ) .
mlst and sequencing of the rtxa amplicon were performed directly on the joint fluid sample from the child who had arthritis and on the recovered pharyngeal isolates .
all carriage isolates and the arthritis strain belonged to mlst 25 and shared rtxa allele 1 , which is among the most common genotypes involved in joint and bone infections in france ( 26,35 ) .
it should be pointed out that , despite the increased sensitivity of naaas , when evaluating the efficacy of prophylactic antibacterial drug administration for eradicating k. kingae from colonized children , cultures have the obvious advantage of detecting living bacteria , whereas the viability of k. kingae organisms in pcr - positive / culture - negative specimens is questionable . in addition
detecting invasive k. kingae disease in patients , searching for asymptomatic but colonized children is crucial in the investigation of an outbreak in a daycare facility to assess the full extent of the contagion , identify attendees at risk for clinical disease , and evaluate the effects of prophylactic antibacterial drugs . because of the high density of the resident bacterial flora and the relatively slow growth of k. kingae , detecting the organism in pharyngeal cultures is difficult .
a differential and selective medium consisting of blood agar with 2 mg / ml of vancomycin ( bav medium ) added has been developed to improve recovery of k. kingae from respiratory cultures ( 36 ) .
this formulation facilitates recognition of -hemolytic k. kingae colonies by inhibiting growth of competitive gram - positive bacteria . in a blinded evaluation ,
the bav medium detected 43 ( 97.7% ) of 44 pharyngeal cultures positive for the organism ; 10 ( 22.7% ) positive cultures were identified on routine blood - agar plates ( p<0.001 ) ( 36 ) . if the original bav formulation ( 36 ) or a similar medium ( 23 ) had not been used , and a chocolate - based agar substituted ( in which the faint ring of hemolysis surrounding k. kingae colonies could not be recognized ) , carriers of the organism might not have been detected among 27 asymptomatic attendees of a daycare facility where a cluster of invasive disease occurred ( 37 )
the bacteriologic identification of k. kingae relies on the following : typical gram stain results , showing pairs or short chains of plump , gram - negative bacilli with tapered ends ; -hemolysis ; pitting of the agar surface ; failure to grow on macconkey medium ; weak oxidase activity and a negative catalase reaction ; production of acid from glucose ; and , with rare exceptions , production of acid maltose ( 8,9 ) .
however , k. kingae tends to retain crystal violet dye and , therefore , it may appear to be gram - positive , and laboratories unfamiliar with its cultural and staining features may misidentify the bacterium altogether or dismiss invasive isolates as culture contaminants .
identification of k. kingae , however , is not difficult , and many commercial instruments and technologies such as vitek 2 ( biomrieux , marcy - letoile , france ) , matrix - assisted laser desorption ionization - time of flight mass spectrometry ( maldi - tof ) , or 16s rdna gene sequencing , correctly identify the organism .
recent studies have demonstrated the presence of a polysaccharide capsule on the surface of k. kingae that exhibits between - strains chemical variation and , most probably , antigenic variation ( 17,18 ) .
these characteristics may assist k. kingae in evading the immune response by facilitating the successive colonization of the host by strains harboring different capsular types .
the polysaccharide capsule probably enables mucosal colonization , the survival of the organism in the bloodstream , and invasion of deep body sites ( 17,18 ) .
in addition , all k. kingae strains produce and secrete a potent repeats - in - toxin ( rtx ) that exhibits a wide range of cytotoxic activity and is particularly deleterious to macrophage - like cells , leukocytes , and synovial cells and , to a lesser degree , to respiratory epithelial cells that improve the organism s chances of surviving in the host and of invading skeletal tissues ( 12,19 ) .
similar to meningococci , k. kingae is carried on the oropharyngeal epithelium ( 20,21 ) , and the colonized mucosa is the portal of entry of the organism to the bloodstream from which it may disseminate to 3 areas for which the bacterium shows particular tropism : joints , bones , or the endocardium ( 22,23 ) .
damage to the upper respiratory surfaces by previous or concurrent viral infections or stomatitis appears to facilitate bloodstream invasion by k. kingae ( 16 ) .
k. kingae isolates show remarkable genomic diversity and , to date , 37 multilocus sequence typing ( mlst ) and 74 pulsed field gel electrophoresis ( pfge ) clones have been identified ( 2426 ) .
carried k. kingae organisms differ in their invasive capabilities ( 27 ) , and simultaneous carriage of 1 genotype is unusual ( 28 ) . whereas some strains , which are frequently isolated from healthy carriers , are seldom if ever detected in patients with clinical disease , others are rarely carried asymptomatically , but are responsible for a high proportion of invasive disease ( 24,27,28 ) .
however , a few strains appear to possess an optimal balance between transmissibility and invasiveness and are common among healthy carriers and among infected patients ( 24,27,28 ) .
a recent study has found that certain virulent k. kingae clones , characterized by a distinct combination of pfge and mlst profiles , are substantially associated with bacteremia with no focal infection , skeletal system invasion , or endocarditis , which suggests biological specialization for invading specific host tissues ( 25 ) .
most young children in whom an invasive k. kingae disease developed have been otherwise healthy . in contrast , children > 4 years of age and adults who become infected frequently have underlying conditions such as congenital heart diseases , chronic renal failure , or a variety of primary immunodeficiencies ( 16 ) . the prevalence rate in healthy children during the second year of life ranges between 10% and 12% ( 7,28 ) , which coincides with the peak attack rate of invasive infections ( 16 ) .
pharyngeal carriage of k. kingae and occurrence of disease before a child is 6 months of age are exceptions , indicating that maternal immunity and limited social contact provide protection ( 7,16,30 ) .
the clinical features of invasive k. kingae infections ( other than endocarditis ) are usually mild , and diagnosis requires a high level of suspicion by clinicians .
many patients with k. kingae associated joint or bone disease are afebrile and blood leukocyte counts , c - reactive protein levels , and erythrocyte sedimentation rates are frequently normal ( 16,31,32 ) .
although bacteremia with no focus is the second most frequent manifestation of invasive k. kingae infections , the condition is probably unsuspected and the diagnosis is likely missed in a large number of cases .
the current guidelines for managing illness in young , febrile children with no apparent source of infection , which use body temperature and leukocyte count as criteria for obtaining blood cultures ( 33 ) , are not sensitive enough for detecting occult k. kingae bacteremia because many infected children have a low - grade fever and may or may not have leukocytosis .
detection of k. kingae infections by culture is highly dependent on the use of adequate laboratory techniques .
the recovery of k. kingae from synovial fluid and bone exudates seeded onto routine solid culture media is suboptimal ( 8) . the yield of cultures can be substantially improved by inoculating clinical specimens into aerobic blood culture vials ( bcvs ) from a variety of commercial systems ( 8) .
attempts to isolate the organism from synovial fluid or bone exudates on routine solid media succeeded in 2 of 25 patients , whereas inoculation of these specimens into aerobic bactec ( becton dickinson , cockeysville , md , usa ) bcvs yielded the organism in all cases after a median incubation of 4 days ( 8) .
when specimens from bcvs determined to be positive by the automated blood culture instrument were subcultured onto a blood agar plate of trypticase soy agar with 5% sheep blood hemoglobin or chocolate agar , k. kingae grew readily , indicating that routine solid media are able to support the nutritional requirements of the organism .
this observation suggests that skeletal system exudates exert a detrimental effect on this fastidious bacterium , and dilution of purulent material in a large volume of broth decreases the concentration of inhibitory factors , improving its recovery ( 8) . in studies conducted in israel and france in which bcvs were routinely inoculated with synovial fluid aspirates from young children who had arthritis , k. kingae was isolated in 48% of the patients with culture - proven disease ( 8,9 ) .
conversely , when bcvs are not used , many k. kingae infections will be overlooked and labeled as culture - negative septic arthritis ( 34 ) . in recent years ,
development of naaas has further improved the diagnosis of k. kingae from skeletal system exudates .
use of this novel technique facilitates detection of difficult - to - culture organisms , enables bacteriologic diagnosis in patients already treated with antibacterial drugs , reduces time to detection , and facilitates precise identification of unusual species ( 1014 ) .
the procedure consists of extraction of bacterial dna from the synovial fluid sample , a pcr amplification step in which primers target the 16s rdna , the 23s rdna , or the rpo genes that are ubiquitous to all bacteria , then sequencing of the amplicon , and comparison of results with those kept in a broad database curator ( such as genbank ) to enable precise species identification .
alternately , the specimen may be subjected to amplification by pcr by using species - specific primers that recognize the most plausible pathogens .
use of naaas has confirmed that in countries where this topic has been studied , k. kingae is the most common etiologic agent of septic arthritis in children <3 years of age .
specific dna sequences , including in cases in which seeding of synovial fluid specimens into bcvs failed to recover the organism and shortened the time required to detect and identify the bacterium from 34 days to < 24 hours ( 1014 ) .
it was , then , natural that this sensitive approach was consequently adopted to study respiratory colonization by k. kingae and its connection to invasive disease . in an outbreak investigated by bidet et al .
, the cause of the skeletal infections was determined by sequential use of real - time pcr targeting the k. kingae
the same method was used to investigate the prevalence of k. kingae among attendees of the index daycare facility .
the organism was recovered by culture in 6 asymptomatic carriers , whereas the naas detected 5 additional carriers ( 35 ) .
mlst and sequencing of the rtxa amplicon were performed directly on the joint fluid sample from the child who had arthritis and on the recovered pharyngeal isolates .
all carriage isolates and the arthritis strain belonged to mlst 25 and shared rtxa allele 1 , which is among the most common genotypes involved in joint and bone infections in france ( 26,35 ) .
it should be pointed out that , despite the increased sensitivity of naaas , when evaluating the efficacy of prophylactic antibacterial drug administration for eradicating k. kingae from colonized children , cultures have the obvious advantage of detecting living bacteria , whereas the viability of k. kingae organisms in pcr - positive / culture - negative specimens is questionable .
in addition detecting invasive k. kingae disease in patients , searching for asymptomatic but colonized children is crucial in the investigation of an outbreak in a daycare facility to assess the full extent of the contagion , identify attendees at risk for clinical disease , and evaluate the effects of prophylactic antibacterial drugs . because of the high density of the resident bacterial flora and the relatively slow growth of k. kingae , detecting the organism in pharyngeal cultures is difficult .
a differential and selective medium consisting of blood agar with 2 mg / ml of vancomycin ( bav medium ) added has been developed to improve recovery of k. kingae from respiratory cultures ( 36 ) .
this formulation facilitates recognition of -hemolytic k. kingae colonies by inhibiting growth of competitive gram - positive bacteria . in a blinded evaluation ,
the bav medium detected 43 ( 97.7% ) of 44 pharyngeal cultures positive for the organism ; 10 ( 22.7% ) positive cultures were identified on routine blood - agar plates ( p<0.001 ) ( 36 ) . if the original bav formulation ( 36 ) or a similar medium ( 23 ) had not been used , and a chocolate - based agar substituted ( in which the faint ring of hemolysis surrounding k. kingae colonies could not be recognized ) , carriers of the organism might not have been detected among 27 asymptomatic attendees of a daycare facility where a cluster of invasive disease occurred ( 37 ) .
the k. kingae colonization rate is substantially enhanced among children in daycare centers . in an 11month longitudinal study , 35 ( 72.9% ) of 48 daycare center attendees carried the organism at least once and an average of 27.5% of the children were colonized at any given time ( 20 ) .
molecular typing of isolates from asymptomatic colonized attendees showed genotypic similarities , indicating person - to - person transmission of the organism in the facility ( 21 ) .
two k. kingae strains represented 28.0% and 46.0% of all isolates , demonstrating that some strains are particularly successful in colonizing mucosa .
children harbored the same strain continuously or intermittently for weeks or months , and then it was replaced by a new strain , showing that carriage is a dynamic process in which there is frequent turnover of colonizing organisms , as observed for other respiratory pathogens ( 21,28 ) . despite the high prevalence of the organism in the daycare center , an invasive k. kingae infection did not develop in any of the attendees in the course of the follow - up period . the link between out - of - home child care and k. kingae carriage
was recently confirmed in a study conducted among 1,277 children < 5 years of age who were referred to a pediatric emergency department ( 7 ) .
daycare attendance was strongly associated with k. kingae carriage after controlling for other variables ( odds ratio 9.66 [ 95% ci 2.9931.15 ] , p<0.001 ) ( 7 ) .
surveillance studies not only have showed that k. kingae organisms colonizing attendees of a given daycare center are frequently identical , but have also demonstrated that carried strains differ between facilities located close together , indicating that each daycare center is like an independent epidemiologic unit ( 35,3739 ) . considering these findings , it is not surprising that clusters of proven and presumptive cases of invasive k. kingae disease have been detected in daycare centers in france ( 35 ) , the united states ( 37,38 ) , and israel ( 39 ) , including 2 recent and still unreported outbreaks ( p. yagupsky , unpub . data ) ( table 1 ) .
a presumptive case was defined as bacteriologically unconfirmed invasive disease , consistent with the clinical features of k. kingae infections , among daycare center attendees 6 months3 years of age , within 1 month of an infection confirmed by culture and/or naaas .
these events have been characterized by the simultaneous or consecutive occurrence of multiple cases of disease that included the entire clinical spectrum of k. kingae infections ( septic arthritis , osteomyelitis , bacteremia , spondylodiscitis , cellulitis , and fatal meningitis complicating endocarditis ) .
the 3 patients in the 2005 cluster in israel ( 39 ) and 4 of the 5 patients reported in france ( 35 ) had bone infections , supporting the concept that certain k. kingae clones exhibit specific tissue tropism ( 25 ) .
sa , septic arthritis ; om , osteomyelitis ; sd , spondylodiscitis ; ce , cellulitis ; ob , occult bacteremia ; en , endocarditis ; mn , meningitis . ; nd , not done ; indicates that no patients manifested this syndrome .
one child had osteomyelitis of the femur and septic arthritis of the contiguous hip joint .
three of the 6 k. kingae illness outbreaks in daycare centers reported during 20032013 were detected in israel , a small country with a population of 8,000,000 inhabitants ( 35,3739 ; p. yagupsky , unpub .
although israel s relatively high annual birthrate compared with western countries ( 18.7 per 1,000 population , israel central bureau of statistics , vs. 10.9 per 1,000 in the european union , demography report 2010 , eurostat ) , and the widespread and early daycare center attendance could partially account for this observation , it seems plausible that similar events occur worldwide but are frequently overlooked .
the epidemiologic investigation of these outbreaks revealed that the k. kingae colonization rate among asymptomatic attendees to the daycare centers where clinical cases were detected was unusually high ( up to 52.9% in a united states cluster , as determined by culture , and 68.8% in france , as demonstrated by a sensitive naaa ) ( table 2 ) , and all pharyngeal isolates detected in the classrooms where disease occurred were genotypically identical and indistinguishable from the patients clinical isolates .
the age of the colonized or infected daycare center attendees coincided with the age of increased susceptibility to k. kingae carriage and disease ( 7,15,16,28 ) ; the organism was detected in the pharyngeal culture of only1 of the caregivers , supporting the hypothesis that healthy adults rarely carry k. kingae ( 29 ) .
as determined by a nucleic acid amplification assay . in all other cases carriage was established by culture on selective media . despite a background carriage rate as high as 5%12% ,
the incidence of invasive k. kingae infections reported in israel was 9.4 per 100,000 children < 5 years of age per year only ( 16 ) , and the calculated annual risk of developing k. kingae osteomyelitis or septic arthritis for young carriers in switzerland was < 1% ( 40 ) . when data from the 6 clusters of invasive disease detected in daycare centers are pooled , a documented or presumptive k. kingae infection developed in 1 in 7 classmates within a 1-month period ( 35,3739 ) , indicating that the outbreak strains combined enhanced colonization fitness , high transmissibility , and remarkable virulence .
the strain responsible for the cluster of osteomyelitis detected in israel in 2005 ( 39 ) belonged to pfge clone k and mlst-6 that ranked second among strains carried in southern israel by healthy jewish children ( 20 ) , and was responsible for the excess of k. kingae illness observed in the jewish population of the region over the previous 2 decades , causing 41.7% of all invasive strains isolated in this ethnic group ( 27 ) .
the clone that caused an outbreak in daycare centers in north carolina , usa ( 37 ) , represented 11.3% of all organisms carried by healthy children in southern israel ( 24 ) and 11.6% of all isolates from patients in israel who had invasive infections ( 27 ) .
the international distribution of these clones indicates that the clusters of disease are frequently caused by highly successful strains that exhibit enhanced capability for local and long - range dissemination ( 26 ) .
to prevent further cases of disease and eradicate the invasive strain , prophylactic antibacterial drugs have been administered to children attending the facilities where clusters of k. kingae were detected .
rifampin was chosen because k. kingae is especially susceptible to that antibacterial drug ( 35 ) .
rifampin is secreted in saliva and reaches high concentrations in the upper respiratory mucosa and has shown efficacy in the eradication of colonization by n. meningitidis and h. influenzae type b , and in disease prevention in daycare centers ( 2 ) .
however , because only partial success was achieved with this drug in the minnesota cluster ( 38 ) , high - dose amoxicillin was added to the regimen in 2 more recent outbreaks ( 37,39 ) .
following administration of antibacterial drugs , a respiratory carriage of the organism decreased , but complete eradication occurred only in the durham , north carolina daycare center ( 37 ) , and new colonization of several attendees by the original strain was later observed ( 35,38,39 ) .
persistence of the organism in the facility was not caused by bacterial resistance to the administered antibacterial drugs , however ( 35,37,39 ) .
similar observations have been made in outbreaks in daycare centers caused by h. influenzae type b and pneumococci ( 2 ) .
poor compliance or failure to administer prophylactic antibacterial drugs to family contacts could have resulted in incomplete suppression of the reservoir and recurrent dissemination of the strain in the facility ( 2 ) . because antibacterial drugs have been relatively ineffective in eradicating k. kingae carriage , the need for antibacterial drug prophylaxis in the setting of a cluster of invasive disease is being disputed ( 40 ) .
notably , however , after administration of antibacterial drugs to the asymptomatic children , no further cases of disease were detected in the affected daycare centers ( 35,3739 ) , even when a few children continue to carry the invasive strain . reducing the bacterial density among colonized children by antibacterial drug administration or an effective immune response induced by prolonged mucosal carriage may have been sufficient to prevent new cases of infection . improved hygiene and institution of other infection control measures
the figure depicts an algorithm aimed to guide the investigation and management of clusters of invasive k. kingae in daycare facilities , including areas of controversy .
suggested algorithm for the investigation and management of clusters of invasive kingella kingae infections in daycare centers .
naaas , nucleic acid amplification assays ; bcvs , blood culture vials ; bav , blood - agar - vancomycin medium .
in recent years , clusters of invasive k. kingae infections among attendees of daycare centers have been reported , although because of the low rate of testing for this pathogen , many events are probably overlooked .
detection of these events requires a high level of clinical suspicion , use of sensitive culture techniques and naaas , and familiarity of the clinical microbiology laboratory with the identification of this elusive pathogen .
the same improved detection methods should be employed for the thorough investigation of these clusters , and recovered k. kingae isolates should be genotyped and compared .
many issues remain unsettled , including whether antibacterial drugs should be administered prophylactically to daycare center contacts of an index case - patient , to young siblings of clinical case - patients , and to carriers , or whether antibacterial drugs should be offered to confirmed carriers only , or limited to children with disease .
if administration of antibacterial drug prophylaxis is decided on , the preferred drug regimen will have to be determined . whether an epidemiologic investigation should be carried out in daycare centers after detection of a single case of disease also remains to be determined . | during the past decade , transmission of the bacterium kingella kingae has caused clusters of serious infections , including osteomyelitis , septic arthritis , bacteremia , endocarditis , and meningitis , among children in daycare centers in the united states , france , and israel .
these events have been characterized by high attack rates of disease and prevalence of the invasive strain among asymptomatic classmates of the respective index patients , suggesting that the causative organisms benefitted from enhanced colonization fitness , high transmissibility , and high virulence .
after prophylactic antibacterial drugs were administered to close contacts of infected children , no further cases of disease were detected in the facilities , although test results showed that some children still carried the bacterium .
increased awareness of this public health problem and use of improved culture methods and sensitive nucleic acid amplification assays for detecting infected children and respiratory carriers are needed to identify and adequately investigate outbreaks of k. kingae disease . |
dopamine receptors and dopamine signaling have been implicated in various neurological and psychiatric disorders including parkinson 's disease , schizophrenia , and drug abuse [ 13 ] .
dopamine receptors are divided into two subfamilies , the gs - coupled d1 and d5 receptors and the gi / o - coupled d2 , d3 , and d4 dopamine receptors that have stimulatory and inhibitory effects on adenylyl cyclase ( ac ) , respectively ( see for a recent review ) .
acute stimulation of d2 dopamine receptors leads to inhibition of ac activity , however , persistent activation of this gi / o - coupled receptor paradoxically results in its enhancement .
this phenomenon , called heterologous sensitization of ac , is also known as camp overshoot , supersensitization , or superactivation of ac .
d2 dopamine receptor - induced heterologous sensitization of cyclic amp signaling has been demonstrated in several cellular systems as well as in animal models and has also been suggested to occur in humans [ 46 ] .
for example , it was observed that repeated administration of the d2 receptor agonist quinpirole enhances ac activity in the caudate putamen , increases creb phosphorylation , and also alters behavior in rodents [ 5 , 6 ] .
although this mode of ac regulation has been recognized for over three decades , the molecular signaling mechanism causing heterologous sensitization of ac is only partially understood , attributed to some extent to differences in ac isoform - specific regulation .
there are nine differentially regulated membrane - bound ac isoforms in mammalian cells [ 4 , 8 ] .
whereas all ac isoforms are stimulated by stimulatory gs , only a subset is inhibited by inhibitory gi , and some ac isoforms are differentially regulated by g [ 4 , 8 ] . here , we studied human adenylyl cyclase type 5 ( ac5 ) that is potently stimulated by gs , inhibited by acute activation of gi , and conditionally activated by g
. ac5 is expressed at high levels in the central nervous system and has been identified as a primary effector of d2 dopamine receptors in the striatum [ 9 , 10 ] .
the aim of the current study was to investigate the role(s ) of heterotrimeric g proteins in d2 receptor - mediated heterologous sensitization of ac5 . by exploring sensitization in cells devoid of endogenous gs
, we were able to examine the ability of gs mutants to support sensitization without interference from endogenous gs .
additionally , this gs - deficient cellular model expresses very low levels of ac5 making them a reasonable model for studies of recombinant ac5 .
heterologous sensitization of ac5 was readily rescued by wild - type gs and by mutants deficient in palmitoylation or g interaction .
we also assessed the role of g and the signalosome in d2 receptor - induced heterologous sensitization of ac5 by sequestering g subunits with arkct - cd8 [ 15 , 16 ] and coexpressing a dominant - negative mutant of the sar1 gtpase .
these experiments revealed that both arkct - cd8 and sar1(h79 g ) attenuated sensitization , suggesting that the components of the signaling complex utilized in heterologous sensitization , presumably ac5 and gs , assemble postsynthesis in the endoplasmic reticulum ( er ) .
together with previous findings , the present data support a model in which gs directly interacts with ac5 .
in contrast , g appears to have an indirect role in heterologous sensitization of ac5 .
the human d2l receptor and ac5 or ac5 were cloned into the dual expression vector pbudce4 ( invitrogen , carlsbad , ca ) creating pbud / hac5 , d2r and pbud/ac5 , d2r .
pcdna3/arkct - cd8 [ 15 , 16 ] and pcdna / vsvg - sar1 ( wild type and h79 g ) were used .
the c3s mutation was created by site - directed mutagenesis , and the fragment containing the iek+ mutations was amplified by pcr .
the resulting constructs , pcdna1/gs - cfp(c3s ) and pcdna1/gs - cfp(iek+ ) were sequenced .
all reagents were purchased from sigma - aldrich ( st . louis , mo ) unless otherwise noted .
gs - deficient murine embryonic fibroblast cells , gnas cells [ 11 , 12 ] , were a gift from dr .
cells were cultured in 50 : 50 mix of f12 : dmem media supplemented with 5% fbs ( hyclone , logan , ut ) , 1% ant - anti ( invitrogen , carlsbad , ca ) in a humidified incubator at 33c with 5% co2 .
approximately 80,000 cells / well were seeded in 24-well plates the day before transient transfection .
dna ( 400 ng pbud / hac5 or ac5 , d2r alone or in combination with 10 ng pcdna / gs - cfp , 300 ng pcdna3/arkct - cd8 , or 300 ng pcdna / vsvg - sar1 ) was mixed with opti - mem and 1 l / well lipofectamine 2000 ( invitrogen , carlsbad , ca ) . the medium was replaced with 200 l / well prewarmed opti - mem , and the dna / lipofectamine mixture was added to the cells .
after 4 hr , culture medium ( 500 l / well ) was added , and the cells were analyzed after 48 hr . for microscopy ,
the amount of pcdna / gs - cfp was increased to 100 ng / well .
the assays were carried out in assay buffer ( ebss supplemented with 0.2% ascorbic acid , 15 mm hepes , and 2% bcs ( hyclone , logan , ut ) , and 500 m ibmx ) with 100 nm forskolin ( tocris bioscience , ellisville , mo ) as noted for 37c for 15 min .
the media was decanted , ice - cold trichloroacetic acid was added , and the lysates were stored at 4c .
data were collected from a minimum of three independent experiments carried out in duplicate and were normalized to either basal or vehicle conditions .
the graphpad prism 5 software ( graphpad software inc . , lajolla , ca ) was used for data and statistical analyses .
the cells were pretreated with 1 m quinpirole or vehicle in assay buffer ( without ibmx ) for 2 hr followed by three washes .
camp was measured as described above for acute camp accumulation , with the addition of 1 m spiperone to block the action of any residual quinpirole .
a 12 bit photometric coolsnap ( roper scientific ) ccd camera mounted on a te-2000 inverted epifluorescence microscope ( nikon instruments inc . ,
images were acquired with the metamorph software ( molecular devices , sunnyvale , ca ) and analyzed using image j ( http://rsbweb.nih.gov/ij/ ) .
our laboratory has previously shown that mutants of canine ac5 that do not interact with gs are deficient in sensitization [ 23 , 24 ] and that d2-mediated heterologous sensitization of ac5 has an absolute requirement for gs .
our present objective was to elucidate the mechanism of gs - dependent heterologous sensitization of human ac5 by utilizing two different gs - cfp mutants ( figure 1(a ) ) .
the c3s substitution eliminates the n - terminal palmitoylation site , which causes gs to mislocalize to the cytosolic fraction .
the iek+ mutant contains a series of substitutions , yielding a g-binding deficient gs that also displays a reduction in palmitoylation .
the gs - cfp constructs were coexpressed with ac5 and d2 . since both c3s and iek+ are deficient in responses to receptor stimulation [ 13 , 21 ] , we used direct stimulation of ac5 with forskolin throughout this study .
basal camp accumulation without any gs was 0.73 0.09 pmol / well , whereas co - expression of gs - cfp increased camp accumulation to 3.12 0.22 pmol / well ( wild - type , wt ) , 4.22 0.06 pmol / well ( c3s ) , and 5.88 0.05 pmol / well ( iek+ ) .
forskolin further stimulated camp with values 2.53-fold over basal levels ( figure 1(b ) ) , indicating that wild - type and both gs mutants functionally couple to ac5 .
next , expression and subcellular localization of the gs - cfp constructs ( in the presence of ac5 and d2 ) were evaluated by fluorescence microscopy ( figure 1(c ) ) .
wild - type gs - cfp showed both plasma membrane and intracellular localization , whereas the c3s and iek+ mutants were predominantly localized intracellularly ( figure 1(c ) ) , consistent with previous reports [ 14 , 21 ] . to assess whether the gs - cfp mutants could rescue heterologous sensitization , cells were pretreated with vehicle or quinpirole followed by camp accumulation .
consistent with our previous report , no sensitization of ac5 was observed in the absence of gs ( figure 1(d ) , ctrl ) .
in contrast , coexpression of wild - type gs - cfp resulted in robust sensitization of ac5 under both basal and forskolin - stimulated conditions ( figure 1(d ) ) .
surprisingly , expression of the gs mutants also significantly rescued heterologous sensitization under basal conditions ( white bars ) and to a lesser degree forskolin - stimulated conditions ( black bars ) . as both mutants are deficient in palmitoylation and membrane localization , neither palmitoylation per se , nor membrane localization of gs appears to be essential for heterologous sensitization of ac5 . although we have established that gs is required for heterologous sensitization , our findings above for the iek+ mutant suggest that direct interactions between gs and g are not critical .
this prompted us to further investigate the role of g in d2 receptor - mediated heterologous sensitization of ac5 .
the c - terminus of -adrenergic kinase or grk2 ( arkct ) has been used to sequester g subunits and inhibit g-mediated signaling events , including heterologous sensitization [ 15 , 25 , 26 ] . in the absence of arkct - cd8 ( membrane bound arkct ) ,
sequestering g blocked sensitization of ac5 , under both basal and forskolin - stimulated conditions , revealing the necessity of g for heterologous sensitization of ac5 ( black bars , figure 2(a ) ) .
in contrast , arkct - cd8 had no substantial effects on acute d2 receptor activation ; quinpirole produced significant inhibition of camp accumulation in the presence of arkct - cd8 ( 77 10% inhibition ; n = 2 , data not shown ) . in an effort to explore the site of action for g-dependent sensitization
this mutant is functional and responds to gs stimulation but is deficient in binding g .
the ac5 mutant displayed significant sensitization that was also blocked by arkct - cd8 ( figure 2(b ) ) , suggesting that n - terminal g binding is not intimately involved in heterologous sensitization of ac5 .
instead , there are clearly additional , unidentified g interaction sites in ac5 that are necessary for heterologous sensitization .
such an assumption is supported by fret and in vitro activation studies of the ac5 deletion mutant as well as studies of ac2 , which possesses multiple motifs for g interaction and regulation that are located in the c1b and c2b domains of ac2 .
other possibilities are that ac5 interacts with endogenous ac isoforms in an ac dimer ( see ) that binds g or that specific g and g subunits or g pairs are involved .
however , it is also possible that the g mechanisms involving sensitization of ac may be indirect .
because sequestering g subunits alters signalosome assembly , we hypothesized that a specific signaling complex could be required for heterologous sensitization of ac5 .
several small gtpases , including sar1 , are involved in signal complex assembly and anterograde protein trafficking .
a series of studies using dominant negative mutants of these gtpases shows that gs and g interact with ac2 during trafficking to the plasma membrane [ 30 , 31 ] and that the gs - ac2 interaction is disrupted by sar1(h79 g ) . to study the possibility that interactions between ac5 and its specific signaling partners play a role , we utilized sar1 and sar1(h79 g ) and noted that coexpression with the dominant negative mutant prevented heterologous sensitization of ac5 ( figure 2(c ) ) .
in contrast , acute d2 receptor - mediated inhibition of ac5 was not significantly blocked in the presence of sar1(h79 g ) ( data not shown ) .
our data are consistent with the findings that sar1(h79 g ) disrupts ac - gs interactions ( as measured by bret or coimmunoprecipitation ) to a larger degree than ac - gi interactions . in contrast
, sar1(h79 g ) did not affect the interactions between ac and g , suggesting that the ac interacts with g at an early step in the endoplasmic reticulum ( er ) , but that the interaction with gs occurs after er export .
the observation that signaling mechanisms of acute activity and heterologous sensitization are differentially affected further supports the hypothesis that heterologous sensitization and acute stimulation are dependent on separate mechanisms and possibly separate signalosome components .
the present data support a complex model of d2 dopamine receptor - induced heterologous sensitization of ac5 where gs appears to directly interact with ac5 . a role for g
was confirmed ; however , our observations suggest an indirect role for g that may be involved during the formation of the sensitization signaling complex . a critical role for ac5 in mediating dopamine responses has been previously demonstrated in ac5 deficient mice , which show impaired responses to d2 receptor activation .
therefore , these results have implications in brain regions where d2 dopamine receptors and ac5 are coexpressed , such as the striatum , which is implicated in drug addiction , motivation , mood , and voluntary movement .
persistent d2 dopamine receptor activation has also been linked to psychiatric disorders ( e.g. , schizophrenia and drug abuse ) and to the adaptive responses associated with drug therapy in parkinson 's disease . enhancing our understanding of the underlying components and mechanisms of heterologous sensitization and regulation of specific ac activity ( in the striatum ) may aid in the development of improved and future therapies for these disorders .
for example , recent studies have identified small molecule inhibitors of g-mediated signaling and ac isoform - specific inhibitors that may offer novel therapeutic strategies for modulating complex cns behaviors involving dopamine receptor signaling . | chronic dopamine receptor activation is implicated in several central nervous system disorders .
although acute activation of gi - coupled d2 dopamine receptors inhibits adenylyl cyclase , persistent activation enhances adenylyl cyclase activity , a phenomenon called heterologous sensitization .
previous work revealed a requirement for gs in d2-induced heterologous sensitization of ac5 . to elucidate the mechanism of gs dependency
, we expressed gs mutants in gs - deficient gnase2/e2
cells .
neither gs - palmitoylation nor gs - g interactions were required for sensitization of ac5 .
moreover , we found that coexpressing arkct - cd8 or sar1(h79 g ) blocked heterologous sensitization .
these studies are consistent with a role for gs - ac5 interactions in sensitization however , g appears to have an indirect role in heterologous sensitization of ac5 , possibly by promoting proper signalosome assembly . |
gestational trophoblastic neoplasia ( gtn ) comprises a spectrum of malignant tumors derived from the trophoblast including the invasive mole ( i m ) , the choriocarcinoma ( ch ) , the placental site trophoblastic tumor ( pstt ) and the epithelioid trophoblastic tumor ( ett ) .
pstt is a rare entity with less than 300 cases reported and accounts for 12% of gtn .
however , a wide range of other presenting symptoms has been reported such as abdominal pain , uterine rupture and galactorrhea ( gillespie and hancock , 2009 ) .
this slow - growing tumor tends to stay initially in the uterus but more than 30% of patients have metastatic disease at the time of presentation ( schmid et al . , 2009 ) .
the sites of metastases include the peritoneum , the lung , the liver and the brain .
pstt may follow any gestational event , normal or pathological pregnancy . in a series of 55 cases ( baergen et al . , 2006 ) , the diagnosis was made within an interval of 34 months following a previous gestation , mainly affecting women of childbearing age .
the prognosis of pstt depends on the disease 's extent ( schmid et al . , 2009 ; baergen et al . , 2006 ;
partial myometrectomy may be performed to preserve fertility ( saso et al . , 2012 ) .
the prognosis drops rapidly when metastatic disease is present . for advanced - stage diseases , a combination of surgery and polychemotherapy
we report the case of a patient who develops a recurrence of an ignored pstt at the vaginal vault diagnosed 8 years after her last pregnancy and 4 years after a hysterectomy for benign condition .
a 46-year - old woman , gravida 4 , para 3 , presented to her gynecologist complaining of abdominal pain and purulent vaginal discharge .
the last one was 8 years prior to her presentation and a tubal ligation was performed .
four years later , she underwent a total hysterectomy for recurrent and infected hematocolpos in semi - urgent clinical conditions with no further histological or radiological preoperative work - up .
histological report showed an incomplete healing of the uterine cesarean scar and a high - grade cervical intraepithelial neoplasia .
clinical and ultrasound examination revealed an ovarian mass consistent with an adnexal abscess of the left ovary and an indurated vaginal vault .
the infectious episode resolved promptly but the vaginal biopsy revealed an unexpected vaginal invasive squamous cell carcinoma .
clinical examination , pelvic mri and pet - ct identified suspicious bilateral multilocular ovarian cysts and a 2 cm mass limited to the vaginal vault without parametrial extension .
the patient underwent resection of the vaginal lesion , bilateral salpingo - oophorectomy and pelvic lymphadenectomy .
the macroscopic examination of the vaginal tumor reveals an ill - defined nodular growth measuring 3.5 3.5 3.8 cm .
the mass is pearly white color with areas of hemorrhage ( fig . 1 , a ) .
on microscopic examination , the tumor consists of a pleomorphic population of polygonal intermediate trophoblast cells with amphophilic to clear cytoplasm .
these cells have often a single irregular highly atypical nucleus with focally prominent nucleoli , but some are multinucleate , resembling to syncytiotrophoblastic giant cells ( fig . 1 , b&c ) . mitotic figures ( 2/10 high power field ) , extensive necrosis , hemorrhage and vascular invasion are observed .
the staining of hcg , hpl and inhibine is only focally positive while the staining of placental alkaline phosphatase ( plap ) is more diffuse .
the staining of ckae1/ae3 and ck7 is positive and the staining of ck5/6 and p63 is negative .
the pathological diagnosis of pstt is proposed and confirmed by the expert panel of pathologists of the belgian referent center of gtd .
, the pathology slides of the hysterectomy performed 4 years previously were reviewed and showed no sign of pstt .
the serum hcg concentration was elevated to 41.0 ui / l the day after the surgery .
considering that surgery remains the preferred treatment for patients with localized pstt , that total surgical resection of the unique lesion was achieved and that pstt tends to be resistant to chemotherapy , observation has been elected .
after a follow - up of 10 months , the patient is still in clinical , biological ( -hcg ) and radiological ( pelvic mri ) remission .
as shown in this case report , clinical and pathological presentation of pstt can be extremely variable .
it can be challenging to distinguish pstt from other forms of gtn or nontrophoblastic tumors .
the review of the pathology by experts in this field is of paramount importance as demonstrated by the experience from several european referral centers .
in contrast to other forms of gtn , pstt arises from the implantation - site intermediate trophoblast . given that hcg
is produced by syncytiotrophoblasts , the serum hcg levels in pstt do not correlate with the tumor burden neither with the malignant behavior and thus have no predictive value ( hassadia et al . , 2005 ) .
furthermore , the hcg - free - subunit is the main form of hcg in pstt , as reported recently by cole et al . , and could be used to distinguish pstt from other trophoblastic neoplasia and nontrophoblastic tumors that produce hcg ( cole et al . , 2006 ) .
the classical immunophenotypic pattern of pstt in comparison of choriocarcinoma and nontrophoblastic tumor is illustrated in table 1 ( mazur , 2005 ) .
however , as observed in the present case , there is some overlapping between diagnostic criteria for pstt and choriocarcinoma .
information about optimal management strategy is restricted because the condition is rare and the natural course varies widely .
the revised 2000 figo staging system ( oncology , 2002 ) has a prognostic and therapeutic usefulness for gtn .
indeed , studies show that prognostic factors of the gtn staging system do not apply accurately to pstt .
the figo anatomical staging system is the strongest predictor of patient outcome presenting with pstt ( schmid et al . , 2009 ; baergen et al . ,
a retrospective review of 62 patients with pstt treated over a 30-year period indicates that the probability of 10-year overall survival for those with stage i , ii or iii / iv disease was respectively 90 , 52 and 49% .
this further supports the importance of an early diagnosis ( schmid et al . , 2009 ) .
other factors associated with an unfavorable outcome include patient 's age ( older than 35 years ) , prolonged intervals from previous pregnancy ( over 48 months ) , prior term pregnancy , high hcg level ( over 1000 miu / ml ) , tumor size , depth of myometrial invasion , tumor necrosis , high mitotic count ( over 5 per 10 hpf ) and clear cytoplasm .
it is interesting to note that the prognostic factors are derived from pathological analysis of the uterus and therefore are not fully applicable for the present case .
( 2009 ) report that the prognosis of patients developing recurrent or refractory disease is poor and that only four ( 22% ) patients achieved long - term survival beyond 60 months . to our knowledge ,
they recommend adjuvant chemotherapy for patients who have risk factors of recurrence or persistent raised postoperative serum hcg level or both .
patients with stage ii , iii or iv pstt are usually managed with combined surgery and chemotherapy .
there is no definitive evidence of benefit of adjuvant chemotherapy for stage ii disease . given that pstt is less chemotherapy responsive than other types of gtn , polychemotherapy is recommended .
although there is no conclusive difference between platinum - based versus non - platinum based regimens , a platinum - containing multidrug chemotherapy is generally preferred such as ep / ema ( schmid et al . , 2009 ) .
gynecologists confronted with vaginal lesion must consider besides primary vaginal tumors , secondary vaginal implants even if the patient underwent a hysterectomy . indeed , 84% of vaginal cancers are metastases and arise not only from gtn but also from other gynecologic malignancies , melanoma , hypernephroma , bladder cancer , breast cancer and colorectal adenocarcinoma .
any gynecologist who has to deal with pstt faces a challenge not only in terms of diagnosis but also in terms of therapy .
although several authors try to establish management strategy , a management algorithm remains controversial because of the lack of data due to the low incidence of the disease and its unpredictable course .
patients should ideally be referred to a center with expertise in gestational trophoblastic disease to assure their optimal care .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
| highlightsrare case of pstt limited to the vagina presenting eight years after last pregnancy and four years after hysterectomydifferential diagnosis with other vaginal tumors can be challenging but it is critical because behavior and management are different.stage-adapted management is proposed and surgery is the mainstay treatment for localized disease . |
the most common symptoms leading to a diagnosis are dysmenorrhoea , pelvic pain , and reduced fertility .
it is a very prevalent disease affecting up to 10% of the reproductive - aged female population .
currently , the most widely accepted theory is the implantation theory : retrograde menstruation can result in viable endometrial cells and fragments entering the peritoneal cavity and once attached , they promote a chronic pelvic inflammatory response .
retrograde menstruation however can not explain all cases , as endometriotic lesions have been identified in diverse locations such as the brain .
it is broadly accepted however that most of the ectopic lesions can be separated into three main regions : ( i ) ovarian , ( ii ) rectovaginal septum ( rvs ) , and ( iii ) peritoneum .
biochemical and pathological differences between the lesions found in these locations have led to suggestions that endometriosis may represent a collection of related but distinct conditions .
it is possible that the variability between these distinct but related lesions is what contributes to the enigmatic nature of the disease .
endometriotics lesions that are established at ectopic sites secrete chemokines which attract macrophages into the peritoneal cavity , further stimulating the inflammatory response and release of cytokines .
significantly increased numbers of activated macrophages have been identified in the peritoneal fluid of women with endometriosis , as has an increased concentration of various chemokines and cytokines .
significantly elevated levels of epithelial neutrophil - activating peptide ( ena-78 ) , monocyte chemotactic protein ( mcp-1 ) , interleukin ( il)-8 , tumor necrosis factor ( tnf)- , il-6 [ 14 , 15 ] , and regulated on activation normal t cell expressed and secreted ( rantes ) have all been found in the peritoneal fluid of patients with endometriosis . underlining the inflammatory nature of the condition
is the fact that tnf , ena-78 , and il-6 [ 18 , 19 ] are also elevated in the serum of women with endometriosis .
less data is however available on the inflammatory response of the lesion itself and whether there is variability based on the type or lesion location .
a difference in the production of specific cytokines may provide an insight into the inflammatory activity of lesions that grow in different locations . in order to gain a better understanding of this complex disease and the differences that can occur between various lesions , this study investigated the production of several chemokines and cytokines in matching eutopic endometrial and ectopic endometriotic tissue and compared their gene expression levels in the three most common presentations of the disease .
laparoscopic surgery was performed for the investigation of pelvic pain or infertility , and any endometriotic lesions identified were removed and their location was noted . where possible , an endometrial biopsy was also collected using a soft curette ( pipelle - de - cornier , laboratoire ccd , france ) . all tissue collected during the surgery
was stored in rnalater ( invitrogen life technologies , zug , switzerland ) at 80c until further use .
exclusion criteria for the study included prior or current infections , liver dysfunction , or the use of hormonal treatments , including any hormonal contraceptive or gonadotropin releasing hormone analogues ( gnrha ) within the past 3 months .
institutional review board approval was obtained from the ethical committee prior to the commencement of the study . after the informed consent was obtained and exclusion criteria were satisfied , we collected eutopic endometrial biopsies from 17 patients .
a single matching ectopic lesion was collected from 15 women , two lesions were collected from another , and three lesions in the final case , resulting in 20 ectopic lesions with matching eutopic samples .
the primary indication for surgery was dysmenorrhea for ten of these women , pelvic pain for four women , and infertility for the remaining three .
the average age of the patients was 32.94 1.454 , range 2441 , and the body mass index ( bmi ) was 23.39 0.914 , range 18.9033.10 . for the further comparison of the mrna expression across ectopic sites
additional lesions were collected from another 23 patients to make a total of 40 patients .
a single lesion was collected from 34 patients , two lesions were collected from five patients , and three lesions were collected from one patient , resulting in a total of 47 ectopic endometriotic lesions . in some cases the isolated mrna was insufficient to determine the concentration of all genes of interest and as such n values are included with each mean and sem .
the primary indication for surgery was dysmenorrhea for 17 women , pelvic pain for another 14 , and idiopathic infertility for the remaining nine .
the average age was 35.58 1.265 , range 2258 , and the bmi was 23.79
no significant difference in either age or bmi was observed in the three groups based on lesion location .
approximately 30 mg of tissue from both the eutopic endometrial biopsies and ectopic endometriotic lesions was excised and homogenized in the fastprep 120 tissue homogenizer ( 30 seconds at 4.0 m / sec ) in cell lysis buffer ( qiagen , dsseldorf , germany ) .
rna isolation was performed with the rnaeasy minikit ( qiagen ) and after isolation the turbodnase kit ( ambion , life technologies , zug , switzerland ) was used for genomic dnase digestion .
one microgram of the total rna was reverse transcribed in a 25 l reaction volume with the moloney murine leukemia virus ( mmlv ) reverse transcriptase ( promega , dbendorf , switzerland ) and random primers .
the resulting cdna was diluted 1 : 20 and the absence of genomic dna was confirmed with a reverse transcriptase control . the quantitative real time polymerase chain reaction ( qpcr ) was performed with the sybr green fast advance master mix ( qiagen ) and a rotor - gene rg 2000 ( corbett research , nsw , australia ) , under the following conditions , 95c for 5 min , followed by 40 cycles of 95c for 5 second , and 60c for 10 seconds .
specificity of the reaction was confirmed via melt curve analysis and the product size was confirmed on a 4% agarose gel .
the genbank accession number and the primer sequences for all genes examined by qpcr are shown in table 1 .
the most stable reference genes and the optimal combination to provide minimal variability were selected via the genorm software program and a geometric mean of the four reference genes selected was used to normalise the expression of the genes of interest for both the eutopic and ectopic tissue .
the reaction efficiency of each assay was determined via linear regression and the fold change calculated with the qbaseplus software ( biogazelle , zwijnaarde , belgium ) .
the difference between the matched eutopic and ectopic mrna expression at different locations and the difference between mrna in different ectopic locations were determined by a one - way analysis of variance ( anova ) test with a post hoc bonferroni 's multiple comparisons test between selected groups .
all values are presented as mean sem and all statistical analysis was performed with graphpad prism 5.0 and significance was set at a value of p < 0.05 .
for the chemokines a one - way anova test confirmed a significant variation between the mrna concentrations of the ectopic endometriotic tissue with eutopic endometrial tissue for ena-78 ( p = 0.0039 ) and rantes ( p = 0.0490 ) , but not for mcp-1 ( p = 0.1251 ) or il-8 ( p = 0.7991 ) ( figure 1 ) .
a bonferroni 's multiple comparisons test was performed to compare the mean of each location against the eutopic mean .
no significant difference was observed for mcp-1 mrna expression between the eutopic tissue ( 0.107 0.015 , n = 17 ) and the ovarian lesions ( 2.751 1.943 , n = 8 , p < 0.05 ) , the peritoneal ( 0.590 0.167 , n = 7 , p < 0.01 ) or the rvs ( 1.865 0.712 , n
= 4 , p < 0.01 ) lesions ( figure 1(a ) ) . for ena-78
there was a significantly stronger expression in the rvs lesions ( 5.905 3.569 , n = 4 , p < 0.01 ) compared to the eutopic tissue ( 0.613 0.250 , n = 17 ) , but no difference was observed in lesions from either the ovaries ( 0.811 0.290 , n = 8) , or the peritoneum ( 1.444 0.504 , n = 7 ) ( figure 1(b ) ) . for il-8
there was no significant variation in the mrna expression in either the peritoneum ( 0.396 0.114 , n = 8) , the ovarian ( 0.409 0.084 , n = 8) , or the rvs ( 1.574 0.385 , n
= 5 ) compared to the eutopic tissue ( 3.979 3.337 , n = 20 ) ( figure 1(c ) ) .
a significantly higher expression of rantes mrna was observed in the rvs ( 0.582 0.264 , n = 5 ,
p < 0.05 ) compared to the eutopic tissue ( 0.239 0.0432 , n = 17 ) , but not in either the peritoneum ( 0.220 0.030 , n
= 5 ) or the ovarian tissue ( 0.190 0.045 , n = 8) ( figure 1(d ) ) . for the inflammatory cytokines a one - way anova test confirmed a significant variation between the mrna concentrations in the eutopic tissue with the mrna concentration in the ectopic tissue for tnf ( p = 0.0014 ) and il-6 ( p < 0.0001 ) ( figure 2 ) .
a post hoc bonferroni 's multiple comparisons test indicated that tnf mrna expression in both the peritoneal ( 1.939 0.667 , n = 8 , p < 0.05 ) and the rvs ( 3.128 1.608 , n = 4 , p < 0.01 ) samples was significantly higher than that observed for their matching eutopic tissue ( 0.444 0.106 , n = 17 ) , although no difference was observed with the ovarian lesions ( 0.291 0.034 , n = 8) ( figure 2(a ) ) . for il-6
there was a significantly higher expression in the rvs region ( 9.308 3.714 , n = 5 , p < 0.0001 ) , but not the ovaries ( 0.689 0.237 ,
n = 7 ) or the peritoneal region ( 0.667 0.237 , n = 7 ) compared to the eutopic tissue ( 0.152 0.091 , n = 17 ) ( figure 2(b ) ) . a significant variation was observed between the mrna expression of tnf ( p = 0.0265 ) and il-6 ( p < 0.0001 ) , amongst the endometriotic lesions from different locations .
a post - hoc bonferroni 's multiple comparisons test indicated that the tnf mrna expression in the rvs ( 2.590 1.357 , n
= 5 ) was significantly higher than in the ovarian lesions ( 0.813 0.144 , n = 24 , p < 0.05 ) , but not in the peritoneal lesions ( 1.711 0.460 , n = 12 ) .
= 6 ) was significantly higher than the expression in both the ovaries ( 1.260 0.323 , n = 24 , p < 0.0001 ) and the peritoneum ( 1.211 0.400 , n = 13 , p < 0.0001 ) ( figure 3 ) .
in contrast no significant difference in mrna expression was observed for any of the four chemokines examined in this study .
= 5 ) was not significantly higher than either the ovarian ( 1.393 0.632 , n = 25 ) or the peritoneal samples ( 0.814 0.215 , n = 13 ) , which was also the case for ena-78 ( peritoneal ; 1.497 0.465 , n = 13 , ovarian ; 2.988 1.429 , n = 25 , rvs ; 4.822 2.969 , n = 5 ) , il-8 ( peritoneum ; 1.548 1.188 , n = 13 , ovaries ; 1.352 0.471 , n = 25 , rvs ; 2.017 0.543 , n = 6 ) , and rantes ( peritoneal ; 0.288 0.064 , n = 11 , ovarian ; 0.364 0.054 , n = 22 , rvs ; 0.528 0.222 , n = 6 ) ( figure 3 ) .
the study showed that the mrna expression of the chemokines ena-78 and rantes , as well as the inflammatory cytokines tnf and il-6 , was significantly increased in the ectopic lesion compared to those in the matched eutopic tissue in women with endometriosis .
for il-6 , ena-78 , and rantes this increase was most significant in the rvs region , whereas for tnf , it was in both the peritoneal lesions and the rvs lesions .
in addition , when compared across lesion locations il-6 was the most highly expressed in the rvs region compared to either the ovaries or the peritoneum .
the results suggest therefore that different inflammatory proteins have separate roles in different lesions and understanding these roles may help to specifically target certain presentations of endometriosis .
in addition , the increased production of many of these proteins by the rvs lesions provides some molecular evidence towards the notion that lesions developing in the rvs are strongly inflammatory . increased expression of chemokines by ectopic endometrial implants
chemokines secreted by the ectopic lesions stimulate the infiltration of macrophages that further contribute to the development of the disease . in this study
we found a significant increase in the expression of rantes and ena-78 in the rvs lesions compared to the matching eutopic tissue .
rantes production by ectopic lesions recruits leukocytes , which then in turn stimulates rantes production creating a feedback loop .
previous studies support this result as rantes correlates with deep infiltrating endometriosis ( die ) , which is most commonly found in the rvs . ena-78
may also play a significant role in the pathogenesis of endometriosis via the activation of macrophages and the adhesion of endometriotic cells to the underlying tissue .
previous studies have shown that both endometrial epithelial and stromal cells produce significant amounts of ena-78 , which is stimulated by il-1 , although this is the first evidence to indicate a significant upregulation in production of ena-78 by rvs lesions .
il-8 has strong chemotactic properties for neutrophils and t lymphocytes and is a potent angiogenic agent .
while numerous studies have shown an upregulation of il-8 in the peritoneal fluid of women with endometriosis [ 29 , 30 ] the source is not clear .
an increase in peritoneal macrophages may be responsible for a higher concentration of il-8 , as would an increased production of il-8 by the endometriotic lesions themselves .. previous evidence shows that both cultured epithelial and stromal endometrial and endometriotic cells produce il-8 [ 26 , 27 ] , although one study found that the ectopic tissue produced less il-8 than the eutopic tissue .
another study on cultured epithelial and stromal cells showed that il-8 secretion is increased after exposure to il-1 .
the lack of a significant difference for il-8 in this study may be a reflection of the need to stimulate il-8 production in endometriotic tissue .
tnf mrna expression was also significantly up - regulated in both the rvs and the peritoneal lesions compared to those in their matching eutopic tissue .
the primary function of tnf is to initiate a cascade of other cytokines that can further stimulate a proinflammatory response . in endometriosis
it correlates with both the stage of the disease , and the menstrual pain reported .
consistent with its early role in the inflammatory cycle it also stimulates cytokines , such as il-6 .
the increased expression of tnf is consistent with an important role for this cytokine in the early pathogenesis of endometriosis that may be common for different types of lesions .
the fact that il-6 is only significantly higher in rvs lesions may suggest that the inflammatory pathway between these two lesions may diverge prior to this point .
il-6 is a multifunctional cytokine that can stimulate cell proliferation and angiogenesis and is hormonally regulated .
different studies have shown both an increase [ 14 , 15 ] or no change in the peritoneal fluid of women with endometriosis compared to women without .
il-6 production has previously been identified in ectopic lesions , however results differ as to whether there is a change in production once the tissue becomes pathological .
some studies have found no significant difference between eutopic endometrium and endometriotic tissue from ovarian endometriosis , whereas others with ovarian endometriosis only , or non - detailed locations , have shown significant increases .
furthermore , an in vitro study from endometrial stromal cells isolated from chocolate ovarian cysts showed a significant ability to produce il-6 with production comparable to that of peritoneal macrophages .
none of these previous studies however have addressed the production of il-6 in lesions from different locations .
a limitation of this study that should be mentioned is the small number of samples available for the rvs region .
this is primarily due to the strict exclusion criteria for this study . as evidence indicates that the use of gnrha can have an effect on the cytokine concentrations in the peritoneal fluid [ 41 , 42 ] we excluded all samples from women with previous gnrha use in the last 3 months . as women with rvs lesions are more likely to experience painful symptoms and to have previously sought treatment for endometriosis
, a large proportion of women presenting to our tertiary care facility with rvs lesions had previous gnrha or contraceptive use and thus were excluded from the study . however , although we only had a small number of samples , the ability to use matched eutopic and ectopic samples and our strict exclusion criteria should provide more weight to these results .
further studies with more samples should be performed to confirm our findings of differential cytokine production from lesions from different locations . in conclusion , this study gives new insights in the production of chemokines and cytokines in endometriotic lesions from different locations and our results support the supposition that the rvs lesions are an intensely inflammatory form of endometriotic lesions .
assessing lesions from different locations uniquely may be vital in understanding the pathological changes of the disease and potentially for their mode of treatment . | endometriosis is characterised by the growth of ectopic lesions at multiple locations outside the uterine cavity and may be considered a collection of distinct but related conditions .
the exact aetiology of endometriosis is still not clear although a role for inflammation is increasingly accepted .
we therefore investigated the inflammatory activity of eutopic tissue and that of the matching ectopic lesions from different locations by measuring the genetic expression of inflammatory chemokines and cytokines .
the gene expression in matching eutopic and ectopic tissue was compared , as was the gene expression in lesions from different locations .
a significantly higher mrna expression of the chemokines ena-78 and rantes and the cytokines il-6 and tnf was observed in endometriotic lesions of the rectovaginal septum ( rvs ) compared to that of matching eutopic tissue .
comparisons across lesion locations showed a significantly higher expression of il-6 and tnf in the rvs compared to lesions from either the ovaries or the peritoneum .
these results show that the production of some inflammatory chemokines and cytokines is significantly increased in the ectopic endometrial tissue compared to matching eutopic tissue .
furthermore , il-6 and tnf are produced in significantly higher quantities in rvs lesions compared to other lesions . |
there are basically three variants of primary localized cutaneous amyloidosis ( plca ) : macular , papular ( lichenoid ) and nodular forms [ 1 , 2 , 3 ] . in macular and papular plca , cytokeratins serve as the amyloid precursors , and these variants are limited to the dermis [ 1 , 2 , 3 , 4 ] . in contrast , the rare type of nodular plca originates from immunoglobulin light chains ; it may extend to the subcutaneous tissue , and blood vessel infiltration can also be observed [ 1 , 2 , 3 , 5 ] .
furthermore , nodular plca can originate from systemic amyloidoses or progress to systemic disease . here
, we present a rare case of nodular plca , which resembled a giant tumor on the head .
a 79-year - old man presented with a large tumor on the left side of his head ( fig .
the waxy tumor exhibited a sulcated , mostly yellowish and reddish surface , and in some areas hemorrhages .
a punch biopsy showed amorphous eosinophilic material in the dermis , so that we suspected a giant amyloid tumor [ 1 , 2 , 4 ] .
histology showed that nearly the entire dermis was filled with amorphous eosinophilic material ( fig .
immunohistochemistry was negative for cytokeratin , but showed that the entire dermis and parts of the subcutis were filled with amyloid consisting of both kappa and lambda immunoglobulin light chains ( fig .
, we performed fine - needle abdominal fat aspiration , and the samples showed only minimal traces of amyloid , thus no signs of systemic amyloidosis .
therefore , the diagnosis was primary localized cutaneous nodular light chain amyloidosis . at 1-year follow - up
the diagnosis of plca requires histological analysis of a skin specimen . in order to discriminate the amyloid precursors in plca , immunohistochemistry with antibodies against cytokeratin and immunoglobulin light chains ( lambda and kappa subtypes )
it is best to also use electron microscopic work - up of a sample fixed in karnofsky 's fixative ( glutaraldehyde ) . as amyloid fibrils are quite stable
, electron microscopy can also be done from formalin - fixed samples ; however , image quality will be reduced with this standard fixative . the typical amyloid fibrils with diameters of 710
nodular plca can often be successfully treated surgically , e.g. via curettage , excision or ablative laser therapy ( co2 lasers etc . ) .
patients should regularly be screened for progression to systemic disease as this may occur in 550% of cases .
to do so , a full history and physical examination along with an electrocardiogram , complete blood count , serum creatinine level , serum liverassociated enzymes levels , serum protein electrophoresis and urine protein electrophoresis should be preformed .
it has also been suggested that an abdominal fat biopsy ( easy access for screening ) be performed to rule out systemic disease . to our knowledge , this is the most extensive case of nodular plca reported in the literature .
| a 79-year - old man presented with a large tumor on the left side of his head , which had grown over 5 years .
regional lymph nodes were impalpable and computed tomography revealed no signs of bone infiltration .
histology showed that the entire dermis was filled with amorphous eosinophilic material .
immunohistochemistry was negative for cytokeratin , but showed that the dermis and parts of the subcutis were filled with amyloid consisting of immunoglobulin light chains .
there were no signs of paraproteinemia or underlying plasmocytoma . in electron microscopy , the typical amyloid fibrils were found .
the tumor was completely removed via curettage . at 1-year follow - up
, the patient was doing fine with no signs of relapse or systemic disease . |
the book of judith is a deuterocanonical book of the septuagint and catholic and eastern orthodox christian old testament of the bible .
it narrates the story of the beautiful jewish widow judith who saved her city of bethulia from the siege of holofernes , a general of the assyrian king nebuchadnezzar . according to the story in the manuscript , after a banquet at which the general had been drinking , the heroine entered holofernes tent and then seduced and killed him .
she beheaded him and presented his head to his fellow citizens ( judith 10:13 ) .
many artists have immortalized the scene of the decapitation , with the heroin holding the sword on the neck of holofernes whilst lying on the bed , and the scene with judith carrying the head of the general as a trophy beside his headless body , often assisted by her maidservant . a systematic reappraisal of the works representing the bloody scene of judith and holofernes has revealed 24 portraits in which judith is depicted with variable degrees of thyroid gland enlargement .
however , there are no spectroscopic or radiographic images available or any written documents attesting the intention of the painters .
therefore , the presence of the goiter can only be judged by an inspection of the portraits .
twenty - four portraits constitute the subject of this review , with particular focus on the neck of the models .
the works representing the story of judith and holofernes were depicted by 21 painters of various nationalities belonging to the renaissance , mannerist , baroque , and later artistic movements and were included in the present investigation . for the purpose of illustration ,
the 24 depictions of judith in which the goiter is visible are combined as panels [ figures 14 ] .
( a ) judith with the head of holofernes ( c. 1575 ) , by jan massys , oil on panel , 115 cm 80.5 cm , ( from the royal museum of fine arts , antwerp , belgium ) ( b ) judith with the head of holofernes ( c. 1550 - 75 ) , by vincent sellaer , oil on panel , 60.5 cm 44.4 cm , ( from the davis museum and cultural center , wellesley , massachusetts , usa ) ( c ) judith and holofernes ( 1540 ) , by jan sanders van hemessen , oil on panel , 99.1 cm 77.2 cm , ( from the art institute , chicago , illinois , usa ) ( d ) judith with the head of holofernes ( c. 1540 - 50 ) , by girolamo da carpi ( girolamo sellari ) , oil on panel , ( from the alte pinakothek , munich , germany ) ( e ) judith ( c. 1548 - 51 ) , by lambert sustris , oil on canvas , 95 cm 113 cm , ( from the palais des beaux - arts de lille , france ) ( f ) judith with the head of holofernes ( c. 1562 ) , by lorenzo sabatini , oil on canvas , 110 cm 85 cm , ( from the banca del monte di bologna e ravenna , bologna , italy ) ( a ) judith and her maidservant with the head of holofernes ( c. 1520 - 30 ) , by guido reni , oil on canvas , 234 cm 150.3 cm , ( from galleria spada , rome , italy ) ( b ) judith and holofernes ( a copy from original of guido reni ) ( 1625 ) , by carlo maratta , oil on canvas , 110 cm 85 cm , ( from the musei capitolini , rome , italy ) ( c ) judith and holofernes ( c. 1610 - 40 ) , by sigismondo coccapani , oil on canvas , 99 cm 76.2 cm , ( from private collection ) ( d ) judith with the head of holofernes ( c. 1605 - 10 ) , by giuseppe cesari aka cavalier darpino , oil on canvas , 48 cm 61.3 cm , ( from the berkeley art museum , ucla , berkeley , california , usa ) ( e ) judith and holofernes ( c. 1600 - 20 ) , by antiveduto grammatica , oil on canvas , 190 cm 159 cm , ( from the derby museum and art gallery , derby , england , uk ) ( f ) judith with the head of holofernes ( c. 1610 - 15 ) , by carlo saraceni , oil on canvas , 90 cm 79 cm , ( from the kunsthistorisches museum , vienna , austria ) ( a ) judith and her maidservant ( 1608 - 9 ) , by orazio gentileschi , oil on canvas , 160 cm 136 cm , ( from the nasjonalgalleriet , oslo , norway ) ( b ) judith and her maidservant ( 1610 - 12 ) , by orazio gentileschi , oil on canvas , ( unknown location ) ( c ) judith and her maidservant ( 1613 - 4 ) , by artemisia gentileschi , oil on canvas , 114 cm 93.5 cm , ( from the palazzo pitti , florence , italy ) ( d ) judith and her maidservant with the head of holofernes ( c. 1623 - 5 ) , by artemisia gentileschi , oil on canvas , 184 cm 141.6 cm , ( from the detroit institute of arts , detroit , michigan , usa ) ( e ) judith slaying holofernes ( c. 1614 - 20 ) , by artemisia gentileschi , oil on canvas , 158.8 cm 125.5 cm , ( from the museo capodimonte , naples , italy ) ( f ) judith ( 1678 ) , by eglon van der neer , oil on oak , 32 cm 24.6 cm , ( from the national gallery , london , uk ) ( a ) judith and her maidservant with the head of holofernes ( c. 1710 ) , by giovanni antonio pellegrini , oil on canvas , 124.7 cm 102 cm , ( from the barber institute of fine arts , university of birmingham , birmingham , england , uk ) ( b ) judith with the head of holofernes ( c. 1730 ) , by giuseppe marchesi , oil on canvas , 149 cm 116.5 cm , ( from the pinacoteca nazionale , bologna , italy ) ( c ) judith and holofernes ( c. 1730 ) , by giulia lama , oil on canvas , 107 cm 155 cm , ( from the gallerie dellaccademia , venice , italy ) ( d ) judith and her maidservant with the head of holofernes ( unknown ) , by venetian school , oil on canvas , ( unknown location ) ( e ) judith with the head of holofernes ( 1695 ) , by giovanni gioseffo dal sole , oil on canvas , 109 cm 90 cm , ( from the minneapolis institute of arts , minnesota , usa ) ( f ) judith ( c. 1840 ) , by august riedel , oil on canvas , 131 cm 96 cm , ( from the neue pinakothek , munich , germany )
goiter was depicted very frequently in works from countries in which iodine deficiency was endemic , and it was only sparsely portrayed by painters living in areas where goiter was not common . the present investigation aimed to shed further light on the presence of goiter in the works of painters from different regions depicting the beheading of holophernes by judith .
it seems unrealistic that the artists inadvertently depicted the goiter unaware of the underlying pathological condition because of their high reputation
. however , there may be many other reasons as to why these painters included thyroid enlargement in their depiction of the subjects . from the renaissance era onward ,
painters have been in pursuit of life - like portraits , which led them to study the exact anatomy of the human body .
indeed , because the neck is one of the visible areas of the body , it became an important focus of attention as well as a decorative element , almost as important as the face .
it is difficult to explain the inclusion of goiter in many portraits of judith based on the geographic origin of the models , the artists being from various areas , not all with iodine - deficiency .
it seems restrictive to suppose that the integration of the goiter was only part of the renaissance tendency toward a more realistic and precise representation of the subjects or that it was a peculiar hallmark of a precise artistic movement .
indeed , several painters across centuries included in this investigation belong to different artistic waves .
there is no reason why the painters should have used models affected by a swelling of the neck linked to autoimmune postpartum thyroiditis or familial thyroid dyshormonogenesis to immortalize the scene of judith .
moreover , the hypothesis that the goitrous gland was portrayed to arouse pity or revulsion , to mock the represented subject , to present an biographical self - portrait feature , or to indicate a lower social status is not likely in the thematic depictions of judith .
an analysis of the evolution of the figures of judith in the history of art may be useful for our purpose .
indeed , in the early christian era , judith was depicted as a cast woman considerably far from being sexual or violent .
it is from the renaissance era onward that the figure of judith took on an interestingly different meaning for many artists and scholars , and painters began to portray judith with a sexualized femininity , sometimes contradictorily combined with masculine aggression . during the baroque period ,
judith became a widespread subject of almost all the painters , and her character was portrayed as more overbearing and violent and idealized as a threatening character to the artist and viewer .
judith 's legend was used as a warning against the captivating dangers of beauty .
she became an icon of female power after the renaissance , and although she was a legendary figure , she was mentioned in 1639 as one of the virtuous women in the masterwork of van beverwijck about the superiority of women to men .
judith is described as exceedingly beautiful in the old testament ( judith 8:7 ) , and it is mentioned that the lord increased her beauty , so that she appeared to all men 's eyes incomparably lovely ( judith 10:4 ) . there is no doubt that the inclusion of goiter in the paintings herein is a stylistic representation . indeed , apart from the almost accurate documentation of goiter by the artists , the sitters in our investigation failed to show further signs or symptoms related to hyperthyroidism or hypothyroidism .
the figures 1a2c and f , 3d and f , 4b f show a low - to moderate - grade swelling of the neck without any other physical signs of thyroid disease . the painters may have depicted a slight enlargement of the neck as a stylistic hallmark and
they may have done this with the purpose of creating an idealized beauty with a more attractive neck .
it is also possible that some of the artists were fascinated by strange physiognomies . in figures 2d and e , 3a
c and e , 4a , a large extended goiter is presented , which may have been symbolic of a more masculine and stronger beauty rather than a delicate and graceful beauty .
it would be difficult to say whether the artists intentionally added a large goiter to highlight aggressiveness and irritability , because a simple inspection did not reveal any sign of graves disease .
on the contrary , the swelling in the eye pouch with slight periorbital and submandibular edema seen in figures 1e and f , 2d , 3b and c , 4c and e may be a forced interpretation of hypothyroidism .
it is likely that we perceive as overweight at the present time what the artists used to perceive as beauty at that time . in all the paintings , the swollen neck , when combined with the rest of the body ,
perfectly reflect the qualities that the painters wanted judith to embody from the renaissance period courage , ( dangerous ) female power , seduction , beauty and eroticism , physical strength , cunning , and intelligence .
judith was a legendary hebrew heroine who saved the jewish people from the armies of the assyrian general holofernes .
her depiction changed from early christianity to a recurrent model of civic virtue in the early renaissance , which was often mixed contradictorily with sexual , feminist , and ironic ( captivating danger of beauty ) themes . throughout this evolution
, painters have used her story to highlight the dangerous power and style of women , reflecting the contemporary ambivalence toward the seductive wiles she used against holofernes .
they tried to represent her courage and determination in plotting and beheading a drunk and besotted general .
we can not entirely reject the claim that the artists used a few affected models for judith 's portraits .
nonetheless , the inclusion of a slight thyroid enlargement may be most likely a stylistic hallmark that represents an idealized female beauty with balanced proportioning of the neck and a graceful quality to the body .
the large extended goiter was probably depicted by the artists as a symbol of powerful masculine body and her courage , and at the same time , it probably also reflected the better anatomic accuracy and knowledge developed in that period .
| judith was a legendary hebrew heroine who beheaded the general holofernes and saved the children of israel from destruction by the assyrian army . in the book of judith , which is still present in the catholic and orthodox christian bibles ,
judith is presented as an illustrious woman who defeated the enemy using her virtue and fortitude .
the present investigation has revealed 24 portraits in which judith has been depicted with variable grades of thyroid gland enlargement on the scene where she decapitates holofernes .
there is no doubt that the integration of a slight thyroid enlargement in the paintings is a stylistic hallmark that portrays an idealized female beauty with a balanced neck and graceful body .
the large extended goiter was probably depicted by the artists as a symbol of a powerful masculine body and her courage , and at the same time , it probably also reflects better anatomic accuracy and knowledge of artists from that period . |
patterning of the nervous system requires neurons to extend axons to targets that are often at great distances from their cell bodies .
polarity information can be provided to growing axons by gradients of attractive or repulsive guidance cues that stimulate different signal transduction pathways in growth cones to guide them up or down the gradient , respectively .
previously known guidance cues include unc-129/bmp , unc-6/netrins , slits , ephrins and semaphorins ( reviewed in 1 ) .
unc-129 encodes a bmp - like tgf- superfamily member identified in c. elegans as a suppressor of dorsal migration of the touch receptor axons induced by ectopic expression of unc-5 in these cells2,3 .
unc-129 is expressed by dorsal body muscles and is graded opposite to unc-6 , which is expressed by cells within the ventral nerve cord ( vnc)3 . as cells and axons are repelled from ventral sources of unc-6/netrin they migrate down the unc-6 gradient and simultaneously migrate toward higher concentrations of unc-129 .
unc-129 influences most of the same migrations that are regulated by unc-6/netrin signaling including the migration of the distal tip cell ( dtc ) and the guidance of da , db , vd , dd , and sdq motoraxons35 .
defects in the migration of these cells and axons are not observed in mutants of the type i ( sma-6 and daf-1 ) or type ii ( daf-4 ) tgf- serine / threonine kinase receptors 3 .
unc-129 therefore acts via a novel signaling mechanism to regulate ventral to dorsal axon guidance and cell migration .
navigation using a gradient requires that cells respond appropriately to graded external cues at both high ( short range guidance ) and low ( long range guidance ) levels of the cue , but it is unknown how sensitivity to the cue is regulated as the cell or cell extension ( e.g. , axon growth cone ) moves up or down the gradient . in c. elegans , unc-6 mediated repulsion of motoraxons and dtcs involves two distinct unc-5 signaling pathways one that is dependent on the unc-40 co - receptor ( unc-5+unc-40 signaling ) and one that is independent of unc-40 ( unc-5-alone signaling)4 . in drosophila ,
axons responding to high levels of graded unc-6/netrin require only unc-5-alone signaling , whereas axons responding to low levels require unc-5+unc-40 signaling6 .
this suggests that unc-40 can enhance growth cone sensitivity to low concentrations of unc-6 , raising the possibility that as a growth cone moves down the unc-6 gradient ( e.g. , by repulsion ) it could enhance its sensitivity to unc-6/netrin by increasing unc-5+unc-40 and decreasing unc-5-alone function .
the data presented here show that unc-129 promotes unc-5+unc-40 signaling and suggest that unc-129 also inhibits unc-5-alone signaling through direct interaction with the unc-5 receptor .
thus we propose that unc-129 functions to increase the sensitivity of the growth cone to continuously lower concentrations of unc-6/netrin encountered as the growth cone moves down the unc-6 gradient by interacting with unc-5 to enhance
unc-129/tgf- regulates the guided migration of cells and axons along the dorso - ventral axis .
unc-129 mutants display an uncoordinated phenotype resulting from defects in the proper ventral to dorsal migration of motor axons .
mutants in the classical tgf- serine / threonine kinase receptors and downstream smads have not been reported to display uncoordinated phenotypes or axon guidance defects .
daf-4 is the only tgf- type ii serine / threonine kinase receptor in c. elegans , and as such , all tgf- signaling is expected to require daf-4 .
daf-4(m63 ) mutants did not display defects in axon guidance , demonstrating that the axon guidance defects observed in unc-129 mutants are not the result of a decrease in tgf- signaling . furthermore , double mutants of daf-4(m63 ) , daf-1(m40 ) or sma-6(wk7 ) null alleles with unc-129(ev554 ) did not inhibit axon guidance defects in the da , db motoraxons observed in unc-129 mutants , demonstrating that unc-129 does not act as a negative regulator of the canonical tgf- signaling pathways ( supplemental fig .
therefore unc-129 affects axon and cell migrations through a mechanism that does not involve activation or inactivation of classical tgf- receptor signaling .
mutants share several cell migration and cell association phenotypes with mutants of the unc-6/netrin signaling pathway .
these include defects in ventral to dorsal motor axon guidance , ventral to dorsal dtc migrations3 and separation of sensory rays in the male tail7 ( see below ) . to examine whether unc-129 might act in the unc-6/netrin pathway , we looked for enhancement of null alleles of netrin - signaling components by unc-129 mutations
. predicted null alleles of unc-5 and unc-6 have nearly fully penetrant motor - axon guidance defects , precluding a quantitative analysis of the effects of unc-129 mutation in these genetic backgrounds .
however , unc-129(ev554 ) and unc-40(e1430 ) nulls have similar , incompletely penetrant guidance defects in the da and db motor neurons ( fig .
we found that unc-129(ev554 ) ; unc-40(e1430 ) doubles are not enhanced for da and db motor axon guidance defects indicating that unc-129 and unc-40 act in the same signaling pathway to regulate motor axon guidance ( fig .
unc-5+unc-40 signaling is required to guide migrations at a distance ( i.e. , at the low end of the predicted unc-6/netrin gradient ) .
unc-5+unc-40 signaling is therefore predicted to be more sensitive to lower levels of unc-6 than
in order to test whether these two unc-5 dependent signaling pathways were similarly specialized to short and long - range migration in the c. elegans motor neurons , we asked whether axons in unc-5 mutants were misguided earlier ( i.e. , closer to ventral sources of unc-6 ) in their migration than those of unc-40 and unc-129 mutants ( figure 1c and d ) .
in fact , misguided axons in unc-5(e53 ) mutants frequently migrate longitudinally along the ventral muscle band or along the lateral seam cells ( 42% ) , suggesting defects in short range migration .
this occurs only rarely in unc-40(e1430 ) or unc-129(ev554 ) mutants ( 7% and 5% respectively ) ( fig .
1d ) . instead , misguided axons in unc-40(e1430 ) and unc-129(ev554 ) animals migrate mainly along the dorsal muscle band , suggesting that only long range migration is interrupted .
we examined axon guidance defects in animals that were doubly mutant for unc-5(e53 ) and unc-129(ev554 ) and found that the severity of axon guidance errors is not worse than in unc-5(e53 ) mutants , demonstrating that unc-129 functions within the unc-5 signaling pathway to regulate axon guidance ( fig .
together with the inability of the unc-129(ev554 ) mutation to enhance unc-40(e1430 ) defects , these data demonstrate that unc-129 functions in unc-5+unc-40 signaling to promote long - range axon repulsion from unc-6 .
if unc-6/netrin uses two different signaling mechanisms to regulate long range ( unc-5+unc-40 ) and short range ( unc-5-alone ) migration in c. elegans , then axons that begin their guided migrations at lower concentrations of the unc-6 gradient ( e.g. , lateral neurons ) are predicted to have a greater requirement for
unc-5+unc-40 signaling than axons that begin migrating at higher concentrations of unc-6 present ventrally ( e.g. , motor axons ) .
avm / pvm and alm / plm cell bodies are located laterally and extend axons that do not initially encounter the high concentrations of unc-6 present ventrally .
ectopic expression of unc-5 in these neurons ( using a mec7p::unc-5(+ ) transgene array ) results in unc-6-dependent dorsal re - routing of their axons8 .
if the model is correct , these migrations should be guided largely through activation of the
unc-5+unc-40 signaling pathway , since they occur at a distance from ventral sources of unc-6 . in support of this , and our finding that unc-129 is required for unc-5+unc-40 signaling , the unc-129 , unc-40 and unc-6 null mutations each caused greater than 90% suppression of these dorsally re - routed migrations2 suggesting that
unc-5 alone signaling has a lesser role in guiding axons that begin migrating at lower concentrations of the unc-6 gradient .
together , these results suggest that unc-129 and unc-5+unc-40 act together to promote increased sensitivity to low levels of graded unc-6 required for long range repulsive guidance . furthermore , the requirement for unc-129 to promote this signaling in c. elegans raises the possibility that in drosophila the ability of only specific axons to mount a long - range repulsive response may be dependent on the exposure of these cells to an unc-129/bmp- like molecule .
because unc-6 signaling through unc-5 and unc-40 is required for the proper ventral to dorsal migration of the distal tip cells ( dtcs)4 , we examined the role of unc-129 in dtc migration .
a single dtc is present at the leading edge of both anterior and posterior hermaphrodite gonad arms and each undergoes mirror - image 3 phase migrations that form the u - shaped bi - lobed gonad ( fig . 2a ) .
the two dtcs start out at the ventral midbody . in the first phase of migration , they migrate longitudinally away from each other along the ventral body wall muscle band . in the second phase ,
the dtcs turn and migrate across the lateral epidermis to the dorsal muscle band . in the third and final phase of migration the dtcs turn on the dorsal side and migrate back toward the mid - body , stopping when they reach the dorsal mid - body region .
unc-5 , and unc-6 mutants frequently fail to initiate the ventral to dorsal phase 2 migration and execute the phase 3 migration on the ventral rather than the dorsal side of the animal with normal timing ( ref 4 and fig .
the failure to execute the ventral to dorsal migration is also observed , although less frequently , in unc-40(e1430 ) mutants4 .
in addition , the ability of unc-40(e1430 ) null mutants to enhance dtc defects in unc-5(e53 ) mutants demonstrates that unc-40 also functions in an unc-5 independent manner ( unc-40-alone signaling ) in the dtc9 . this additional function of unc-40 may explain why unc-40 mutants display dtc defects while unc-129 mutants do not .
unc-129 mutations enhance the phase 2 dtc defects of the unc-5(e152 ) hypomorph , demonstrating that unc-129 , and by inference
unc-5+unc-40 signaling , does play a role , although probably a minor one , in this migration ( ref 9 and fig .
interestingly , unc-40(e1430 ) mutants display weakly penetrant defects in which the dtc begins to migrate dorsally but does not complete the ventral to dorsal migration and instead returns to the ventral side ( possibly because it prefers ventral muscle as a substratum for migration ) to complete its third phase migration9 .
this phenotype may be a manifestation of a defect in long - range migration . to determine if unc-129 could function in the unc-5+unc-40 pathway for initiating dtc migrations , we analyzed double mutants of the unc-129(ev554 ) null mutation with predicted unc-5 , unc-6 , and unc-40 null alleles , which cause incompletely penetrant phase 2 dtc migration defects4 .
double mutants of unc-129(ev554 ) and null alleles of either unc-5 , or unc-40 fail to show any enhancement of dtc defects ( fig .
the inability of unc-129(ev554 ) to enhance unc-40(e1430 ) , or unc-5(e53 ) for phase 2 dtc defects suggests that , as for motor - axon migrations , unc-129 functions within the unc-5+unc-40 signaling pathway to regulate dtc migration .
unc-129 mutations were shown previously to enhance male ray fusions ( erf phenotype ) in hypomorphic mab-20(bx61 ) mutants7 .
we found that unc-5(e53 ) and unc-40(e1430 ) genetic null mutations were also able to significantly enhance ray fusions in mab-20(bx61 ) males ( fig .
3 ) . to determine whether unc-129 also acts in the unc-5+unc-40 signaling pathway in the male tail , we looked for enhancement of the mab-20(bx61);unc-129 double mutant ray fusions by unc-5 or unc-40 mutations ( fig .
mab-20(bx61);unc-40(e1430);unc-129(ev554 ) or mab-20(bx61);unc-5(e53);unc-129(ev554 ) triple mutants were not enhanced for ray fusions relative to mab-20(bx61);unc-129(ev554 ) mutants ( fig .
this demonstrates that in the male tail , as for motor axon guidance and initiation of phase 2 dtc migration , unc-129 is required for
in the course of analyzing male ray fusions , we found that unc-129(ev554);mab-20(bx61 ) double mutants displayed a higher penetrance of fusions in rays 3 and 4 ( fig .
3b- boxed ) , relative to unc-5(e53);unc-129(ev554);mab-20(bx61 ) triple mutants , indicating that in the absence of unc-129 activity , unc-5 functions inappropriately to cause ray fusions .
the inability of unc-40(e1430 ) to suppress these fusions suggests that unc-129 acts to block inappropriate
thus , in preventing male ray fusions , as in motor axon guidance , unc-129 is required in the same pathway as
unc-5-alone signaling to unc-5+unc-40 signaling . substantial support for this idea is provided by the analysis of animals ectopically expressing unc-129 .
similar to unc-129 mutations , ectopic expression of unc-129 in the ventral and dorsal muscles using the myo-3 promoter ( using transgene array evis87a [ myo-3p::unc-129(+ ) ; dpy-20(+ ) ] ) also causes defects in the guidance of the commissural motor axons3 .
in order to further investigate the cause of these defects , we introduced unc-40(e1430 ) into evis87a .
in contrast to the lack of enhancement observed in unc-129(ev554);unc-40(e1430 ) double mutants , unc-40(e1430 ) enhanced evis87a axon guidance defects ( fig .
unc-5+unc-40 signaling , ectopic ventral expression of unc-129 interferes with guidance functions independent of this signaling pathway and given the absence of other signaling pathways most likely does so by interfering with
in contrast to the enhancement of dtc defects in evis87a animals by unc-40(e1430 ) , neither unc-5(e53 ) nor unc-5(ev813 ) null alleles enhanced ( supplemental figure 2s ) .
unc-5-alone signaling , which is precisely the signaling we expect to guide migrations at the highest concentrations of unc-6 present ventrally .
however , the involvement of unc-40-alone signaling in dtc migration9 makes it difficult to assess whether the enhancement of evis87a by unc-40(e1430 ) is due to the loss of
because unc-129 is a secreted protein that is required to promote unc-5+unc-40 signaling while inhibiting unc-5-alone signaling , we hypothesized it might do so by binding directly to unc-5 . in order to test this hypothesis ,
we used a modification of lumier , a luciferase - based assay of protein - protein interactions10 .
briefly , we tagged unc-129 at the amino - terminus of the mature protein with gaussia luciferase ( gluc - unc-129 ) and expressed the fusion protein gluc - unc-129 in 293 t cells .
conditioned media was collected and added exogenously to cells expressing either the unc-5/ha receptor or control cells .
immunoprecipitates of unc-5/ha yielded a 7 fold increase in luciferase activity relative to mock transfected cells ( no receptor ) or cells expressing an equivalent amount of ha - tagged tgf- type i receptor ( fig 5a ) .
thus , unc-129 physically interacts with unc-5 . in order to demonstrate that this is a direct interaction , we examined unc-5 interactions in cross - linking experiments . because purified unc-129 is not available , we labeled mammalian bmp7 with i and tested its interaction with unc-5 or rcm ( a mammalian unc-5 homologue ) .
the inability of bmp7 to bind to unc-40 is consistent with our finding that unc-129 inhibits
unc-5-alone signaling in the absence of unc-40 and suggests that unc-129 interaction with unc-40 is not required for its function .
a saturation curve of bmp7 binding to rcm revealed an affinity of 4 nm for this interaction ( fig .
5c and d ) , which is similar to that reported for other bmp ligands and their classical type i and type ii serine / threonine kinase receptors11,12 .
the ability of bmp7 to bind to both unc-5 and rcm suggests that mammalian bmp ligands share similar binding properties with unc-129 .
interestingly , at least three mouse bmps ( bmp7 , bmp6 , and gdf7 ) are expressed in the spinal cord roof plate while netrin is expressed by the floor plate .
this mimics the unc-129 expression pattern ( dorsal ) relative to unc-6 ( ventral ) in c. elegans .
bmp7 is required to promote commissural axon guidance in the spinal cord , as are the mouse homologs of unc-6/netrin signaling components13 .
however , the analogy to c. elegans axon guidance is unclear since the vertebrate commissural axons migrate toward the source of netrin , rather than away from it , suggesting that bmp7 does not guide these axons via the same mechanism used by unc-129 to guide motor axons in c. elegans .
however , the spinal accessory motorneurons are better candidates for using bmp7 the way c. elegans uses unc-129 since their cell bodies and axons migrate from ventral to dorsal in the developing mammalian spinal cord and require both netrin-1 and dcc / unc-40 to do so14 . when cell or axon growth cone migrations are guided by a graded cue , the concentration of the cue encountered by the moving cell or growth cone as it moves up or down the gradient is constantly changing .
thus , the migrating cell or growth cone faces a potential problem of being able to respond appropriately to the gradient at different concentrations of the cue .
for example , a growth cone responding at low concentrations of a cue gradient should require a more sensitive response than it would if it were responding at high concentrations of the same gradient . in drosophila , growth cones exposed to low levels of unc-6/netrin ( at a distance from the netrin source ) require the unc-5+unc-40 pathway for guidance whereas growth cones exposed to high concentrations of the netrin gradient , signal either largely or solely through the unc-5-alone pathway6 .
this suggests that enhanced sensitivity required to respond to low levels of unc-6/netrin can be acquired by increased signaling through unc-5+unc-40. the inability of all neurons expressing these receptors to mount a long - range response suggests that additional factors like unc-129 are required to promote this long - range guidance at low levels of unc-6/netrin .
we have shown that unc-129 promotes unc-5+unc-40 signaling , which is more sensitive to lower concentrations of unc-6/netrin , while antagonizing unc-5-alone signaling , which is specialized to higher concentrations of unc-6/netrin signaling .
thus , as a growth cone is repelled away from ventral sources of unc-6 , it continually encounters lower levels of unc-6 and higher levels of the oppositely graded unc-129 .
the increased levels of unc-129 would , in turn , increase sensitivity to unc-6 by shifting signaling from unc-5-alone to the more sensitive unc-5+unc-40 signaling pathway .
thus unc-129 would cause an appropriate change in the set point sensitivity of the growth cone as it moves down the unc-6 cue gradient so that the growth cone can still sense and respond to the gradient at unc-6 levels so low they might not be sensed by unc-5 alone .
unc-5-alone to unc-5+unc-40 signaling by binding to unc-5 and blocking its interaction with an unidentified protein ( x in supplemental fig .
alternatively , these two signaling pathways may be restricted to different sub - cellular compartments , and unc-129 may control the trafficking of unc-5 between these compartments .
trafficking of dcc , an unc-40 homologue , to lipid rafts following netrin stimulation has been described15 .
if unc-129 promotes movement of unc-5 into lipid raft compartments , it could put unc-5 into proximity with unc-40 thereby enhancing
unc-5+unc-40 complex formation and function and allow unc-5 and unc-40 to function separately in the absence of unc-129 .
in either case , the effect of unc-129 could occur gradually , as cells and growth cones migrate , creating a balance of unc-5-alone vs. unc-5+unc-40 receptor complexes that is constantly re - adjusted towards unc-5+unc-40 signaling as growth cones move farther away from unc-6 and closer to dorsal sources of unc-129 or may occur at a specific time or landmark encountered during migration .
unc-129/tgf- regulates the guided migration of cells and axons along the dorso - ventral axis .
unc-129 mutants display an uncoordinated phenotype resulting from defects in the proper ventral to dorsal migration of motor axons .
mutants in the classical tgf- serine / threonine kinase receptors and downstream smads have not been reported to display uncoordinated phenotypes or axon guidance defects .
daf-4 is the only tgf- type ii serine / threonine kinase receptor in c. elegans , and as such , all tgf- signaling is expected to require daf-4 .
daf-4(m63 ) mutants did not display defects in axon guidance , demonstrating that the axon guidance defects observed in unc-129 mutants are not the result of a decrease in tgf- signaling . furthermore , double mutants of daf-4(m63 ) , daf-1(m40 ) or sma-6(wk7 ) null alleles with unc-129(ev554 ) did not inhibit axon guidance defects in the da , db motoraxons observed in unc-129 mutants , demonstrating that unc-129 does not act as a negative regulator of the canonical tgf- signaling pathways ( supplemental fig .
therefore unc-129 affects axon and cell migrations through a mechanism that does not involve activation or inactivation of classical tgf- receptor signaling .
mutants share several cell migration and cell association phenotypes with mutants of the unc-6/netrin signaling pathway .
these include defects in ventral to dorsal motor axon guidance , ventral to dorsal dtc migrations3 and separation of sensory rays in the male tail7 ( see below ) . to examine whether unc-129 might act in the unc-6/netrin pathway , we looked for enhancement of null alleles of netrin - signaling components by unc-129 mutations
. predicted null alleles of unc-5 and unc-6 have nearly fully penetrant motor - axon guidance defects , precluding a quantitative analysis of the effects of unc-129 mutation in these genetic backgrounds .
however , unc-129(ev554 ) and unc-40(e1430 ) nulls have similar , incompletely penetrant guidance defects in the da and db motor neurons ( fig .
we found that unc-129(ev554 ) ; unc-40(e1430 ) doubles are not enhanced for da and db motor axon guidance defects indicating that unc-129 and unc-40 act in the same signaling pathway to regulate motor axon guidance ( fig .
unc-5+unc-40 signaling is required to guide migrations at a distance ( i.e. , at the low end of the predicted unc-6/netrin gradient ) .
unc-5+unc-40 signaling is therefore predicted to be more sensitive to lower levels of unc-6 than
in order to test whether these two unc-5 dependent signaling pathways were similarly specialized to short and long - range migration in the c. elegans motor neurons , we asked whether axons in unc-5 mutants were misguided earlier ( i.e. , closer to ventral sources of unc-6 ) in their migration than those of unc-40 and unc-129 mutants ( figure 1c and d ) .
in fact , misguided axons in unc-5(e53 ) mutants frequently migrate longitudinally along the ventral muscle band or along the lateral seam cells ( 42% ) , suggesting defects in short range migration .
this occurs only rarely in unc-40(e1430 ) or unc-129(ev554 ) mutants ( 7% and 5% respectively ) ( fig .
1d ) . instead , misguided axons in unc-40(e1430 ) and unc-129(ev554 ) animals migrate mainly along the dorsal muscle band , suggesting that only long range migration is interrupted .
we examined axon guidance defects in animals that were doubly mutant for unc-5(e53 ) and unc-129(ev554 ) and found that the severity of axon guidance errors is not worse than in unc-5(e53 ) mutants , demonstrating that unc-129 functions within the unc-5 signaling pathway to regulate axon guidance ( fig .
together with the inability of the unc-129(ev554 ) mutation to enhance unc-40(e1430 ) defects , these data demonstrate that unc-129 functions in unc-5+unc-40 signaling to promote long - range axon repulsion from unc-6 .
if unc-6/netrin uses two different signaling mechanisms to regulate long range ( unc-5+unc-40 ) and short range ( unc-5-alone ) migration in c. elegans , then axons that begin their guided migrations at lower concentrations of the unc-6 gradient ( e.g. , lateral neurons ) are predicted to have a greater requirement for
unc-5+unc-40 signaling than axons that begin migrating at higher concentrations of unc-6 present ventrally ( e.g. , motor axons ) .
avm / pvm and alm / plm cell bodies are located laterally and extend axons that do not initially encounter the high concentrations of unc-6 present ventrally .
ectopic expression of unc-5 in these neurons ( using a mec7p::unc-5(+ ) transgene array ) results in unc-6-dependent dorsal re - routing of their axons8 .
if the model is correct , these migrations should be guided largely through activation of the
unc-5+unc-40 signaling pathway , since they occur at a distance from ventral sources of unc-6 . in support of this , and our finding that unc-129 is required for unc-5+unc-40 signaling , the unc-129 , unc-40 and unc-6 null mutations each caused greater than 90% suppression of these dorsally re - routed migrations2 suggesting that
unc-5 alone signaling has a lesser role in guiding axons that begin migrating at lower concentrations of the unc-6 gradient .
together , these results suggest that unc-129 and unc-5+unc-40 act together to promote increased sensitivity to low levels of graded unc-6 required for long range repulsive guidance . furthermore , the requirement for unc-129 to promote this signaling in c. elegans raises the possibility that in drosophila the ability of only specific axons to mount a long - range repulsive response may be dependent on the exposure of these cells to an unc-129/bmp- like molecule .
because unc-6 signaling through unc-5 and unc-40 is required for the proper ventral to dorsal migration of the distal tip cells ( dtcs)4 , we examined the role of unc-129 in dtc migration .
a single dtc is present at the leading edge of both anterior and posterior hermaphrodite gonad arms and each undergoes mirror - image 3 phase migrations that form the u - shaped bi - lobed gonad ( fig . 2a ) .
the two dtcs start out at the ventral midbody . in the first phase of migration , they migrate longitudinally away from each other along the ventral body wall muscle band . in the second phase ,
the dtcs turn and migrate across the lateral epidermis to the dorsal muscle band . in the third and final phase of migration the dtcs turn on the dorsal side and migrate back toward the mid - body , stopping when they reach the dorsal mid - body region .
unc-5 , and unc-6 mutants frequently fail to initiate the ventral to dorsal phase 2 migration and execute the phase 3 migration on the ventral rather than the dorsal side of the animal with normal timing ( ref 4 and fig .
the failure to execute the ventral to dorsal migration is also observed , although less frequently , in unc-40(e1430 ) mutants4 .
in addition , the ability of unc-40(e1430 ) null mutants to enhance dtc defects in unc-5(e53 ) mutants demonstrates that unc-40 also functions in an unc-5 independent manner ( unc-40-alone signaling ) in the dtc9 . this additional function of unc-40 may explain why unc-40 mutants display dtc defects while unc-129 mutants do not .
unc-129 mutations enhance the phase 2 dtc defects of the unc-5(e152 ) hypomorph , demonstrating that unc-129 , and by inference
unc-5+unc-40 signaling , does play a role , although probably a minor one , in this migration ( ref 9 and fig .
interestingly , unc-40(e1430 ) mutants display weakly penetrant defects in which the dtc begins to migrate dorsally but does not complete the ventral to dorsal migration and instead returns to the ventral side ( possibly because it prefers ventral muscle as a substratum for migration ) to complete its third phase migration9 .
this phenotype may be a manifestation of a defect in long - range migration . to determine if unc-129 could function in the unc-5+unc-40 pathway for initiating dtc migrations , we analyzed double mutants of the unc-129(ev554 ) null mutation with predicted unc-5 , unc-6 , and unc-40 null alleles , which cause incompletely penetrant phase 2 dtc migration defects4 .
double mutants of unc-129(ev554 ) and null alleles of either unc-5 , or unc-40 fail to show any enhancement of dtc defects ( fig .
the inability of unc-129(ev554 ) to enhance unc-40(e1430 ) , or unc-5(e53 ) for phase 2 dtc defects suggests that , as for motor - axon migrations , unc-129 functions within the unc-5+unc-40 signaling pathway to regulate dtc migration .
unc-129 mutations were shown previously to enhance male ray fusions ( erf phenotype ) in hypomorphic mab-20(bx61 ) mutants7 .
we found that unc-5(e53 ) and unc-40(e1430 ) genetic null mutations were also able to significantly enhance ray fusions in mab-20(bx61 ) males ( fig .
3 ) . to determine whether unc-129 also acts in the unc-5+unc-40 signaling pathway in the male tail , we looked for enhancement of the mab-20(bx61);unc-129 double mutant ray fusions by unc-5 or unc-40 mutations ( fig .
mab-20(bx61);unc-40(e1430);unc-129(ev554 ) or mab-20(bx61);unc-5(e53);unc-129(ev554 ) triple mutants were not enhanced for ray fusions relative to mab-20(bx61);unc-129(ev554 ) mutants ( fig .
this demonstrates that in the male tail , as for motor axon guidance and initiation of phase 2 dtc migration , unc-129 is required for
in the course of analyzing male ray fusions , we found that unc-129(ev554);mab-20(bx61 ) double mutants displayed a higher penetrance of fusions in rays 3 and 4 ( fig .
3b- boxed ) , relative to unc-5(e53);unc-129(ev554);mab-20(bx61 ) triple mutants , indicating that in the absence of unc-129 activity , unc-5 functions inappropriately to cause ray fusions .
the inability of unc-40(e1430 ) to suppress these fusions suggests that unc-129 acts to block inappropriate
thus , in preventing male ray fusions , as in motor axon guidance , unc-129 is required in the same pathway as
unc-5-alone signaling to unc-5+unc-40 signaling . substantial support for this idea is provided by the analysis of animals ectopically expressing unc-129 .
similar to unc-129 mutations , ectopic expression of unc-129 in the ventral and dorsal muscles using the myo-3 promoter ( using transgene array evis87a [ myo-3p::unc-129(+ ) ; dpy-20(+ ) ] ) also causes defects in the guidance of the commissural motor axons3 . in order to further investigate the cause of these defects , we introduced unc-40(e1430 ) into evis87a .
in contrast to the lack of enhancement observed in unc-129(ev554);unc-40(e1430 ) double mutants , unc-40(e1430 ) enhanced evis87a axon guidance defects ( fig .
unc-5+unc-40 signaling , ectopic ventral expression of unc-129 interferes with guidance functions independent of this signaling pathway and given the absence of other signaling pathways most likely does so by interfering with
in contrast to the enhancement of dtc defects in evis87a animals by unc-40(e1430 ) , neither unc-5(e53 ) nor unc-5(ev813 ) null alleles enhanced ( supplemental figure 2s ) .
unc-5-alone signaling , which is precisely the signaling we expect to guide migrations at the highest concentrations of unc-6 present ventrally .
unc-40-alone signaling in dtc migration9 makes it difficult to assess whether the enhancement of evis87a by unc-40(e1430 ) is due to the loss of unc-40-alone signaling or loss of
because unc-129 is a secreted protein that is required to promote unc-5+unc-40 signaling while inhibiting unc-5-alone signaling , we hypothesized it might do so by binding directly to unc-5 . in order to test this hypothesis
, we used a modification of lumier , a luciferase - based assay of protein - protein interactions10 .
briefly , we tagged unc-129 at the amino - terminus of the mature protein with gaussia luciferase ( gluc - unc-129 ) and expressed the fusion protein gluc - unc-129 in 293 t cells .
conditioned media was collected and added exogenously to cells expressing either the unc-5/ha receptor or control cells . expressed receptors
immunoprecipitates of unc-5/ha yielded a 7 fold increase in luciferase activity relative to mock transfected cells ( no receptor ) or cells expressing an equivalent amount of ha - tagged tgf- type i receptor ( fig 5a ) .
thus , unc-129 physically interacts with unc-5 . in order to demonstrate that this is a direct interaction , we examined unc-5 interactions in cross - linking experiments . because purified unc-129 is not available , we labeled mammalian bmp7 with i and tested its interaction with unc-5 or rcm ( a mammalian unc-5 homologue ) .
the inability of bmp7 to bind to unc-40 is consistent with our finding that unc-129 inhibits
unc-5-alone signaling in the absence of unc-40 and suggests that unc-129 interaction with unc-40 is not required for its function
. a saturation curve of bmp7 binding to rcm revealed an affinity of 4 nm for this interaction ( fig .
5c and d ) , which is similar to that reported for other bmp ligands and their classical type i and type ii serine / threonine kinase receptors11,12 .
the ability of bmp7 to bind to both unc-5 and rcm suggests that mammalian bmp ligands share similar binding properties with unc-129 .
interestingly , at least three mouse bmps ( bmp7 , bmp6 , and gdf7 ) are expressed in the spinal cord roof plate while netrin is expressed by the floor plate .
this mimics the unc-129 expression pattern ( dorsal ) relative to unc-6 ( ventral ) in c. elegans .
bmp7 is required to promote commissural axon guidance in the spinal cord , as are the mouse homologs of unc-6/netrin signaling components13 .
however , the analogy to c. elegans axon guidance is unclear since the vertebrate commissural axons migrate toward the source of netrin , rather than away from it , suggesting that bmp7 does not guide these axons via the same mechanism used by unc-129 to guide motor axons in c. elegans .
however , the spinal accessory motorneurons are better candidates for using bmp7 the way c. elegans uses unc-129 since their cell bodies and axons migrate from ventral to dorsal in the developing mammalian spinal cord and require both netrin-1 and dcc / unc-40 to do so14 . when cell or axon growth cone migrations are guided by a graded cue , the concentration of the cue encountered by the moving cell or growth cone as it moves up or down the gradient is constantly changing .
thus , the migrating cell or growth cone faces a potential problem of being able to respond appropriately to the gradient at different concentrations of the cue .
for example , a growth cone responding at low concentrations of a cue gradient should require a more sensitive response than it would if it were responding at high concentrations of the same gradient . in drosophila , growth cones exposed to low levels of unc-6/netrin ( at a distance from the netrin source ) require the unc-5+unc-40 pathway for guidance whereas growth cones exposed to high concentrations of the netrin gradient , signal either largely or solely through the unc-5-alone pathway6 .
this suggests that enhanced sensitivity required to respond to low levels of unc-6/netrin can be acquired by increased signaling through unc-5+unc-40. the inability of all neurons expressing these receptors to mount a long - range response suggests that additional factors like unc-129 are required to promote this long - range guidance at low levels of unc-6/netrin .
we have shown that unc-129 promotes unc-5+unc-40 signaling , which is more sensitive to lower concentrations of unc-6/netrin , while antagonizing unc-5-alone signaling , which is specialized to higher concentrations of unc-6/netrin signaling .
a unifying model is that unc-129 accomplishes this by converting unc-5-alone signaling to unc-5+unc-40 signaling .
thus , as a growth cone is repelled away from ventral sources of unc-6 , it continually encounters lower levels of unc-6 and higher levels of the oppositely graded unc-129 .
the increased levels of unc-129 would , in turn , increase sensitivity to unc-6 by shifting signaling from unc-5-alone to the more sensitive unc-5+unc-40 signaling pathway .
thus unc-129 would cause an appropriate change in the set point sensitivity of the growth cone as it moves down the unc-6 cue gradient so that the growth cone can still sense and respond to the gradient at unc-6 levels so low they might not be sensed by unc-5 alone .
unc-5-alone to unc-5+unc-40 signaling by binding to unc-5 and blocking its interaction with an unidentified protein ( x in supplemental fig .
alternatively , these two signaling pathways may be restricted to different sub - cellular compartments , and unc-129 may control the trafficking of unc-5 between these compartments .
trafficking of dcc , an unc-40 homologue , to lipid rafts following netrin stimulation has been described15 .
if unc-129 promotes movement of unc-5 into lipid raft compartments , it could put unc-5 into proximity with unc-40 thereby enhancing
unc-5+unc-40 complex formation and function and allow unc-5 and unc-40 to function separately in the absence of unc-129 .
in either case , the effect of unc-129 could occur gradually , as cells and growth cones migrate , creating a balance of unc-5-alone vs. unc-5+unc-40 receptor complexes that is constantly re - adjusted towards unc-5+unc-40 signaling as growth cones move farther away from unc-6 and closer to dorsal sources of unc-129 or may occur at a specific time or landmark encountered during migration .
evis82b or evis82a [ unc-129p::gfp ] were used to visualize the da and db motorneurons3 .
animals were anesthetized with 1 mm levamisole and examined using differential interference contrast ( dic ) optics to visualize the gonad .
da and db motor - axons were scored as defective if they failed to reach the dorsal nerve cord .
the n - terminal signal peptide was replaced with the rcm signal peptide and the c - terminal death domain was replaced with an ha epitope .
a description of the luciferase based binding assay is available in supplemental materials and methods .
, 293 t cells were plated onto poly - l - lysine ( sigma ) plates , transiently transfected using calcium phosphate20 and incubated with 4 nm iodine labeled bmp7 for 4 hours at 4c .
cells were washed , crosslinked with dissuccinimidyl suberate ( dss ) ( pierce ) and lysed in tnte ( 50 mm tris ph 7.4 , 150 mm nacl , 1 mm edta ) containing 0.5% triton x-100 .
immunoprecipitations were performed using anti - ha , 12ca5 ( hybridoma supernatant ) , followed by incubation with protein - g sepharose beads ( pharmacia ) .
beads were washed and boiled in sample buffer then samples were separated on sds page gels and exposed to phosphoimager cassette or western blotted with anti - ha antibodies ( 12ca5 ) . to generate saturation curves ,
increasing amounts of i - bmp7 were added to cos-7 cells expressing rcmha or the control vector .
the n - terminal signal peptide was replaced with the rcm signal peptide and the c - terminal death domain was replaced with an ha epitope .
a description of the luciferase based binding assay is available in supplemental materials and methods .
, 293 t cells were plated onto poly - l - lysine ( sigma ) plates , transiently transfected using calcium phosphate20 and incubated with 4 nm iodine labeled bmp7 for 4 hours at 4c .
cells were washed , crosslinked with dissuccinimidyl suberate ( dss ) ( pierce ) and lysed in tnte ( 50 mm tris ph 7.4 , 150 mm nacl , 1 mm edta ) containing 0.5% triton x-100 .
immunoprecipitations were performed using anti - ha , 12ca5 ( hybridoma supernatant ) , followed by incubation with protein - g sepharose beads ( pharmacia ) .
beads were washed and boiled in sample buffer then samples were separated on sds page gels and exposed to phosphoimager cassette or western blotted with anti - ha antibodies ( 12ca5 ) . to generate saturation curves , increasing amounts of i - bmp7 were added to cos-7 cells expressing rcmha or the control vector .
| summarythe unc-5 receptor mediates axon repulsion from unc-6/netrin through unc-40 dependent ( unc-5+unc-40 ) and independent ( unc-5-alone ) signaling pathways . a requirement for unc-40 dependent
signaling has been shown in long - range repulsion from unc-6/netrin , however , the mechanisms used to regulate distinct unc-5 signaling pathways are poorly understood . here
we demonstrate that the c. elegans tgf- family ligand unc-129 , graded opposite to unc-6/netrin , functions independent of the canonical tgf- receptors to regulate unc-5 cellular responses . we provide evidence that unc-129 facilitates long - range repulsive guidance of unc-6 by enhancing
unc-5+unc-40 signaling at the expense of
unc-5-alone signaling through interaction with the unc-5 receptor .
this increases the set point sensitivity of growth cones to unc-6/netrin as they simultaneously migrate up the unc-129 gradient and down the unc-6 gradient .
similar regulatory interactions between oppositely graded extracellular cues may be a common theme in guided cell and axon migrations . |
generally , in automatic titration procedure based on
flow injection analysis process , the volume of the titrant
solution delivered has been determined considering both flow rate
and time interval elapsed while the titration was run
[ 13 ] . in principle , it has been admitted that the flow rate was constant and the time interval was a very
well - controllable variable [ 4 , 5 ] . in these cases ,
peristaltic pump has been the device widely used to propel titrant solution
[ 68 ] . because pumping pulsation , due to
peristaltic pump , could impair the precision of the delivered
solution volume , a synchronization strategy has been implemented
[ 4 , 9 ] . since peristaltic pump maintain a constant pulsation pattern , small volume of titrant solution , presenting good
precision , could be delivered . nevertheless , to accomplish this
requirement , it is necessary to resort facilities comprising
electronic and software [ 4 , 9 , 10 ] .
furthermore , the aging of the pumping tube could affect flow rate ,
thus this effect could become an error source . in this work
, we intend to develop a device with
ability to deliver small aliquots of titrant solution , determining
also its volume with good precision and accuracy independent of
both time and flow rate
. the usefulness of the device will be
demonstrated , coupling then to an automatic titration module based
on multicommutation process [ 11 ,
12 ] .
an 89.20.9 moll hcl solution was prepared by
dilution from a 1 moll hcl solution .
this solution was standardized
by titration using an 88.70.8 moll naoh solution , which was
previously standardized by titration using a potassium biphthalate
solution and a 0.01% ( w / v ) phenolphthalein solution as indicator .
the equipment set up comprised a pentium iii
microcomputer furnished with an electronic interface card
( pcl-711s , advantech ) , an ipc-4 ismatec peristaltic pump with
tygon pumping tubes , six three - way solenoid valves ( 161t031 , nresearch ) , one solenoid pinch valve normally closed ( 161p011 , nresearch ) , a homemade 12 v regulated power supply
to feed the solenoid valves , a homemade electronic interface to provide the potential difference and current intensity required to drive the solenoid
valves , and a homemade photometric detector .
the control unit comprised one photodiode ( hamamatsu , s1337 - 1010br ) , one phototransistor ( til78 ) wired as an
optic switch , two infrared led ( =930 nm ) , two operational amplifiers ( op07 ) , one
comparator device ( lm339 ) , and one transistor ( bc547 ) .
the assays
comprising the volume meter calibration and photometric titration
were performed employing a software wrote in quick basic 4.5 .
, it is shown a front view of the volume
meter , where we can see that the device is coupled to the titrant
solution reservoir ( tr ) through the solenoid valves v1 and v2 .
the v2 was assembled to permit the container loading ( tc ) with titrant
solution when valve v1 was switched on .
when the container was
filled with titrant solution , the floating device ( b ) interrupted the infrared radiation beam of
the optic switch ( opt ) .
when the electronic interface showed in
figure 2 sensed the interruption of the radiation beam , it
generated the control signal to switch off valve v2 in order to
stop the stream of the titrant solution .
the optic switch ( opt ) comprising an infrared
radiation emitting diode ( =930 nm ) and a phototransistor ( til78 ) , which was attached
in a metallic piece ( 550.4 cm ) with a central lead hole to permit the inserting
of the solution container ( tc ) , thus allowing that the container
was positioned between the phototransistor ( pht ) and the infrared
led .
a hole with a diameter of 1.0 mm was drilled at the orthogonal
position to the lead hole in order to permit that the infrared
beam attained the phototransistor ( pht ) after it crosses the
titrant solution container ( tc ) . as it is depicted in figure 1 , an infrared
radiation emitting diode ( led1 )
was installed at the bottom of the
titrant solution container ( tc ) , thus the radiation beam emitted
by this led was directed towards the photodiode ( pd ) through the
liquid column maintained inside of the container .
the liquid
column behaves as a wave guide and it was observed that its length
affected the intensity of the infrared radiation beam that reaches
the photodiode ( pd ) . the volume meter was developed exploiting
this effect .
the filling of titrant solution container ( tc ) was controlled by mean of the electronic interface showed in figure 2 . prior
to load the container with titration solution ,
the system was adjusted following the steps summarized in
table 1 . maintaining the titrant solution container empty ,
the
signal generated by the optic switch ( opt ) was 3.8 v that was
applied to the no inverting input of the comparator device ( lm339 ,
pin 5 ) .
reference signal applied to the comparator input ( pin 4 )
was adjusted to 3.5 v turning the variable resistor ( 20 k ) wired to it .
under this condition , the output of the comparator device ( pin 2 )
was at a high - state condition and the power - driving device ( pw )
was maintained deactivate , while the control enabling line ( ens )
was at low level ( 0 v ) .
when the software was run , the
microcomputer sent a control signal ( ttl high level ) through
pcl711 interface card to enable the control line ( ens , figure 2 ) . under this condition ,
the input line ( d0 ) of the power - driving device ( pw )
was enabled to switch on valve v1 , thus the titrant solution flowed from its vessel ( tr , figure 1 ) by the gravity acceleration action to fill the titrant container ( tc )
with titrant solution . while the liquid column increased into the
container , the floating device ( b ) was displaced toward the top .
when it crossed the light beam of the optic switch ( opt ) , the
signal sent to the comparator input ( pin 5 ) fall lower than 3.0 v.
under this condition , the output signal of the comparator device
( pin 2 ) was drove to the low - state condition ( 0 v ) , thus disabling
the line input ( d0 ) of the power - driving device ( pw ) , which
switched off valve v1 in order to stop the stream of titrant
solution .
the light emitting diode ( led1 ) installed in the
container bottom ( tc , figure 1 ) was switched off by turning the
variable resistor wired to the base of the transistor ( tr1 , figure 3 ) .
the signal output ( so ) of the operational amplifier ( oa2 ) was adjusted to 0 v by turning the variable resistor ( 20 k ) wired to the
no inverting input of this operational amplifier . afterwards
, the
emission intensity of the led1 was raised up to the output signal
( so ) attained 2000 mv .
this was done turning the variable resistor
coupled to the base of the transistor ( tr1 ) .
the flow diagram of the titration system is shown in
figure 4 . in this configuration ,
all valves are switched off ,
thus all solutions propelled by the peristaltic pump ( pp ) are
directed towards their storing vessels . when valve v2 was switched
on
, the titrant solution flowed through it . aiming to find the
relationship between the solution volumes delivered from the
container ( tc ) and the output signal ( so ) generated by photodiode
( figure 3 ) , a set of assays
the microcomputer
sent , through the output port of the pcl711s interface card , a
control signal to switch on valve v2 .
while solution flowed from
the container ( tc ) , the microcomputer read the variation of the
signal ( so ) through the analog input of the pcl711s interface
card .
when signal variation attained a preset value ( vs ) , valve v2
was switched off in order to stop the solution stream , and the
delivered volume of solution was measured .
assays were performed
programming potentials difference ranging from 50 up to 800 mv .
after calibration stage , the valve v2 was maintained switched on
during a time interval of 2 minutes to empty the titrant solution container .
after calibration , a set of titration assays was
carried out to demonstrate the usefulness of the proposed volume
meter , which was implemented using the flow system showed in
figure 4 , which was designed based on multicommutation process
[ 1012 ] .
titration chamber ( tch ) and photometer detector ( not showed ) were similar to those
employed in previous work . as it is depicted in table 1 , in the standby
condition all valves
are switched off , thus all solutions are
pumped back to their storing vessels ( figure 4 ) . when the
software was started to carry out a titration run , the microcomputer sent a control signal ( ttl high level ) through the enabling line ( ens , figure 2 ) to enable the power - driving
device ( pw , figure 2 ) . under this condition ,
valve v1
was switched on to fill the container ( tc ) with titrant solution .
afterwards ,
valves v5 and v6 were switched on during a time interval of
20 seconds ( step 3 ) to fill the flow lines up to the titration
chamber ( tch ) .
afterwards , valve v7 was switched on during a time
interval of 15 seconds ( step 4 ) to empty the titration chamber .
the
washing of the titration chamber with carrier solution comprised
the steps 5 and 6 ( table 1 ) , which were repeated twice to
assure good cleaning .
following the working sequence showed in table 1
( steps 7 , 8) , valves v5 and v6 were sequentially switched on during
preset time intervals to insert into the titration chamber
aliquots of sample solution ( s ) and of indicator solution ( in ) ,
respectively . the time interval to maintain valve
v6 switched on
was settled at 2.0 seconds , while time for valve v5 was varied from
3.0 up to 8.0 seconds .
afterwards , valves v3 and v4 were switched on
during a time interval of 8.0 to insert 800 l of the carrier solution into the titration chamber . as it is shown in table 1 ( step 10 ) ,
valve v2 was switched on to permit that the titrant solution flowed continuously by the gravity acceleration action toward the titration chamber ( tch ) .
the
monitoring of the signal generated by the photometer was performed
by the microcomputer through the analog input of the pcl711s
interface card .
when a quick transition of the generated signal
occurred , the microcomputer sent a control signal through the
enabling line ( ens , figure 2 ) to switch off valve v2 in order to stop the stream of the titration solution .
afterwards , the microcomputer read
the signal ( s0 ) generated by the photodiode ( pd ) of the volume
meter ( figure 3 ) that was saved as an ascii file .
the volume of
the titrant solution used to find the end of titration was
calculated using this measurement .
when the signal ( s0 ) surpassed a
preset threshold value , which was related to a delivered solution
volume of the titrant solution about 85 l , the
microcomputer halted the stream of the titrant solution and saved
the signal value related with the solution volume that was used at
that moment .
afterwards , the volume meter - filling step was carried
out again and after that the titration run was reestablished in
order to find the end of titration .
the volume meter working principle was based on the
attenuation of the radiation beam intensity , which was affected by
the height of the liquid column inserted between the
electromagnetic radiation source ( led1 , figure 1 ) and the
photodiode ( pd ) .
the value of the potential difference generated
by the photodiode decreased when the gap between the liquid column
top and the photodiode ( pd ) increased .
previous assays performed
using leds with wavelength of 470 nm ( blue ) , 650 nm ( red ) , and 930 nm
( infrared ) showed that better linear range could be obtained with the infrared led . in this sense , the results commented below were obtained employing this component . intending to find the relationship between the volume
of the solution delivered and the potential difference generated
by the photodiode ( pd ) , a set of assays was carried out yielding
the results showed in table 2 .
processing these data , we found
the following linear relationship : e(mv ) = 25.7 7.4 v(l ) , ( r=0.999 ) .
aiming to confirm the linear relationship ,
experiments were carried out at several days .
the assays were done
settling different values of potential difference and no
significant difference was observed .
every day prior to begin the first titration run , the
calibration stage was accomplished in order to find the parameters
related to the linear relationship , which were incorporated in the
control software to permit that the volume of titrant solution was
calculated when titration run was ended . intending to prove the
ability of the device to deliver a preset solution volume ,
a set
of experiments was performed using distilled water and results are
showed in table 3 .
processing these data , a linear relationship
presenting a slope of 0.999 and an intercept tending to 0.0 was achieved .
these results indicated that the system was able to determine the
solution volume processing the potential difference related to
liquid column height .
furthermore , the precision of the delivered
volumes was better than 1.0% ( n=10 ) which was confirmed repeating the experiments
maintaining the preset values . aiming to demonstrate the usefulness of the proposed
volume meter device , photometric titration of hydrochloric acid
with sodium hydroxide solution was performed yielding the results
showed in table 4 .
the volumes of the hcl solution aliquots
were varied from 43 up to 115 l . as we can
see the concentration of the hcl solution found is closed to
expected values . in the assays labeled as 5 and 6 the volumes of the
hcl solution aliquots used were 100 and 115 l ,
respectively , thus the solution container ( tc , figure 1 ) was
filled twice .
analyzing the results , we can see , that in both
cases accuracy and precision were similar to the other ones showed
in table 4 .
the performance of the proposed volume meter shows
that it can be used to deliver small volume with good precision
independent of the flow rate and the time interval .
the
instrumental arrangement is very simple and easy to be replicated
in laboratory with a few little electronic facilities .
the volume meter was successfully applied in the
determination of the titrant solution volume in photometric
titration procedure of hydrochloric acid with sodium hydroxide
solution .
albeit , the operational work range was between 5 and 105 l , the
appropriated combination of software and hardware were able to
refill the titrant solution container while the titration run was
in course .
the volume meter device presented a very good
long - term stability , which was ascertained by working several days
and no significant variation concerning to linear response range ,
precision , and accuracy was observed . | in this work , an automatic device to deliver titrant solution into a titration chamber with the ability to determine the dispensed volume of solution , with good precision independent of both elapsed time and flow rate , is proposed .
a glass tube maintained at the vertical position was employed as a container for the titrant solution .
electronic devices were coupled to the glass tube in order to control its filling with titrant solution , as well as the stepwise solution delivering into the titration chamber .
the detection of the titration end point was performed employing a photometer designed using a green led ( =545 nm ) and a phototransistor .
the titration flow system comprised three - way solenoid valves , which were assembled to allow that the steps comprising the solution container loading and the titration run were carried out automatically .
the device for the solution volume determination was designed employing an infrared led ( =930 nm ) and a photodiode .
when solution volume delivered from proposed device was within the range of 5 to 105 l , a linear relationship ( r = 0.999 ) between the delivered volumes and the generated potential difference was achieved .
the usefulness of the proposed device was proved performing photometric titration of hydrochloric acid solution with a standardized sodium hydroxide solution and using phenolphthalein as an external indicator .
the achieved results presented relative standard deviation of 1.5% . |
thyroid nodules are commonly diagnosed in adults ; however , they are rare cases in pediatrics . depending on the iodine intake ,
they have been ranged from 19 to 35% in adults by ultrasonography ( us ) and from 0.2 to 5.1% in children [ 2 , 3 ] .
cancer risk of a thyroid nodule is significantly greater in children than in adults , as it ranges from 3 to 70% and 5 to 14% [ 2 , 420 ] in regard to children and adults , respectively .
current research suggests that up to 25% of thyroid nodules in children are malignant compared to 5% in adults [ 2 , 21 ] .
the possibility of the wide range of cancers led in the past some experts to recommend that children with nodules should proceed directly to thyroidectomy without fine needle aspiration cytology ( fnac ) or other preoperative testing . also , thyroid cancer is present in children in a clinically more aggressive form as regards metastases and extrathyroidal extension [ 9 , 22 , 23 ] . the epidemiological data differs from one research group to another , likely reflecting methodological differences ( e.g. , tertiary pediatric centers are prone to overestimation of cancer prevalence ) .
iodine deficiency has a well - known and deep effect on the prevalence of thyroid nodules , on the ratio of papillary to follicular carcinomas , and even on the diagnostic power of fnac .
therefore , we analyzed our 20 years ' experience with childhood nodular goiter disease in a moderately iodine - deficient region .
although the analysis was retrospective , our evaluation process has a unique advantage : the clinical research , us , and fnac were performed by the same investigator and the pathological analysis was performed by the same histopathologist during this entire period .
in a 20-year period from 1994 to 2014 all patients ( n = 3,010 ) were enrolled in the study who were aged 20 or less ( group 1 ) and who entered our thyroid outpatient departments for the first time .
if the tsh was under the normal value , ft4 and ft3 test were performed , as well . if the us was not fully intact and echonormal , anti - tpo test was performed .
patients with nodules larger than 1 cm in maximal diameter and those who presented with hypoechogenic discrete lesion at least 5 mm in maximal diameter underwent fnac .
we compared the data of group 1 with those of 3,010 randomly selected patients who were older than 20 ( group 2 ) .
the control patients entered our thyroid outpatient department also for the first time and the evaluation process was the same as described above .
the analysis was retrospective and all clinical examinations , us , and fnacs ( both the aspiration and the microscopic analysis ) were performed by the same physician ( tamas solymosi ) .
we used the following categories in cytological diagnosis : benign , suspicious , malignant , and nondiagnostic .
based on our previous experiences , which are in accordance with the published data of [ 25 , 26 ] , spongiform and central types of mixed nodules share minimal , if any , risk being malignant in adult patients . therefore ,
if the cytological material was deficient in giving a cytological diagnosis and the solid part of the nodule presented neither microcalcification nor irregular vascularization in the event of spongiform and central type cysts in group 2 , that is , in adult patients , we gave a common us - fnac diagnosis as a result , cystic nodule with less than 1% risk of malignancy , and grouped these lesions among benign cases .
however , these lesions were categorized as nondiagnostic cytology in group 1 because of the limited experiences in younger patients .
the histopathological analysis was performed by the same pathologist ( gyula lukacs toth ) during the entire period .
the proportion of intact thyroids was significantly greater in younger compared with adult patients ( p < 0.001 , = 1314.1 ) .
the proportion of autoimmune thyroid diseases occurred significantly more frequently in younger patients than in older patients , too ( 77.5% ( 799/1031 ) and 59.7% ( 1429/2394 ) , group 1 and group 2 , resp . , p <
the ratio of hashimoto 's thyroiditis in euthyroid state to primary hypothyroidism was significantly higher in younger than in adult patients ( 1.56 ( 397 to 255 ) and 0.45 ( 372 to 821 ) , group 1 and group 2 , resp .
the proportion of hyperthyroidism in the case of thyroid dysfunctions was significantly higher among younger patients compared with older patients ( 29.8% ( 108/363 ) and 17.2% ( 171/992 ) , group 1 and group 2 , resp .
the proportion of patients with nodular goiter was significantly lower in younger than in adult patients ( 22.8% ( 235/1031 ) and 39.3% ( 942/2394 ) , group 1 and group 2 , resp .
( p < 0.01 , = 87.6 ) ) . ultrasound characteristics of thyroid nodules are given in table 2 .
we analyzed the occurrence of discrete lesions in euthyroid hashimoto 's patients ( table 3 ) .
significantly more patients had discrete lesions at least 5 mm in maximal diameter in group 2 than in group 1 ( p < 0.01 , = 26.8 ) , and the ratio of true nodules was also significantly higher in group 2 compared with group 1 ( p < 0.01 , = 28.1 ) .
the ratio of adenoma to hyperplastic nodular goiter was significantly higher in younger than in adult patients ( 51 to 17 and 46 to 77 , group 1 and group 2 , resp .
younger patients were characterized by a greater proportion of papillary carcinomas , a greater proportion of follicular variant of papillary carcinomas , and a more aggressive clinical presentation ( see table 4 ) .
significantly higher proportion of nodular goiters was operated on in younger patients than in adults ( 33.2% ( 78/235 ) and 15.2% ( 143/942 ) , group 1 and group 2 , resp .
first , more younger patients were operated with benign cytology because of the decision of the patients ( or their parents ) compared with older patents ( 25% ( 9/36 ) and 9.5% ( 9/95 ) , group 1 and group 2 , resp . ) .
second , the proportion of patients operated on the cytological suspicion because of follicular tumor was higher among younger patients than in adults ( 17.9% ( 14/78 ) and 11.2% ( 16/143 ) , group 1 and group 2 , resp . ) .
the latter was the consequence of the above - mentioned fact , the significantly greater proportion of adenomas among younger compared with adult patients .
similar proportion of histologically diagnosed adenomas and lesions other than adenomas was categorized statistically correctly in a statistical manner in younger and in adult patients .
the diagnostic accuracy in the event of adenomas was significantly worse compared with other lesions 60.5% ( 46/76 ) and 94.9% ( 112/118 ) , adenomas and lesions other than adenoma , respectively .
this led to worse specificity , positive predictive value , and diagnostic accuracy in younger compared with adult patients . regarding the preoperative us diagnosis of nodules in operated hashimoto 's patients , the us diagnosis was correct in 21/23 cases .
a pseudonodule is characterized by the following features : it is multiple and hypoechogenic , is in the range of 1 to 10 mm , and presents an ill - defined , frequently puzzle - like margin .
the thyroid displays other signs of the underlying autoimmune disease in most cases ; it has at least a minimally hypoechogenic and/or inhomogeneous basic echostructure .
two of 7 and 5 of 20 peripheral type cysts proved to be malignant ( group 1 and group 2 , resp . ) .
no carcinoma was found among 66 spongiform or central type mixed solid - cystic nodules .
the ratio of nondiagnostic fnacs was significantly higher in group 1 compared with group 2 ( p < 0.01 , = 7.74 ) .
this is explained by the significantly greater proportion of spongiform and central type cysts categorized as nondiagnostic in younger compared with adult patients ( 12/29 ( 41.4% ) and 5/37 ( 13.5% ) , group 1 and group 2 , resp .
there was a striking and significant difference in the spectrum of thyroid diseases among children and adults in our study .
almost 60% of children referred for an evaluation had intact thyroids , while this proportion was only 12% in adults .
around 75% of children presented with autoimmune thyroid diseases , and 21% with thyroid nodules in diseased patients . these rates were 60% and 40% in adults , respectively .
our results demonstrate that the spectrum of nodular goiter in children living in an iodine - deficient region does not differ from those living in an iodine - sufficient area .
significantly more nodular goiters are malignant and the clinical presentation ( i.e. , the ratio of nonmicrocarcinomas to microcarcinomas , the ratio of papillary carcinomas metastatic to lymph nodes to nonmetastasizing presentations , and the proportion of t4 tumors ) is greater in children than in the case of adults .
the high ratio of the follicular variant of papillary carcinoma once again is in accordance with the previous observations .
significantly smaller proportion of nodules was proved to be malignant in our series compared with most series in the medical literature , 4.5% and 25% [ 2 , 21 ] , our data and data in the literature , respectively . the difference is explained by the well - known goitrous effect of iodine deficiency .
some authors , similarly to us , have found that adenomas are the most frequent benign disease in childhood ; however , others have found hyperplastic nodules as the dominant nonmalignant disease .
the proportion of adenomas among histologically verified nodules ranges from 23.3% to 61.5% [ 10 , 13 , 21 , 27 , 28 ] .
this ratio was 75% in our study which is in accordance with our previous finding : iodine deficiency increases the prevalence of follicular adenoma .
the proportion of adenomas to hyperplastic nodules may have a deep consequence even on the evaluation process : an adenoma can not be cytologically distinguished from a follicular carcinoma ; therefore , the greater proportion of adenomas leads to a greater concern and worse results in fnac diagnosis .
this is even more important in children : the ratio of adenoma to hyperplastic nodule was significantly higher in children compared with adults .
there is a similarly important effect on the entire evaluation process regarding the ratio of follicular carcinomas to papillary carcinomas .
some have found no follicular carcinoma in children [ 10 , 13 , 29 , 30 ] which showed similar results to ours , while other groups have found a ratio of follicular to papillary carcinoma from 3 : 7 to 1 : 12 [ 15 , 21 , 3135 ] . both of the above - mentioned issues , that is , the ratio of hyperplastic nodules to adenomas and the real prevalence of follicular carcinomas , reflect one of the greatest concerns in thyroidology : the correct and reproducible histopathological interpretation of follicular lesions including follicular variant of papillary carcinoma [ 3638 ] .
we may try to compare the data of different authors ; however , it is very challenging to draw unequivocal consequences from the various analyses , unless the issue with the interpretation is resolved . in our opinion
it is worthy to reconsider one of the general dogmas of the evaluation of thyroid patients .
this dogma means that the diagnosis of a follicular tumor clearly leads to an operation .
it may lead ( in our opinion it already led ) to an apparent discrepancy : we state that our goal is to recognize malignant thyroid nodules , but we behave as our goal would be to recognize and to operate on benign tumors , as well . the thyroidologist needs to accept the fact that the cost of recognition of follicular carcinomas involves unnecessary surgeries in the case of a follicular adenoma .
however , despite the increasing number of well - differentiated thyroid carcinomas , follicular thyroid carcinoma is getting diagnosed less and less frequently .
this means we have to pay a high price : 51 children with follicular adenoma were sent to have surgery without anyone gaining positive results from it . if other researchers confirm our data that follicular adenoma occurs significantly more frequently and follicular carcinoma less frequently in children , then a new approach seems to be mandatory in cytologically diagnosed follicular tumors , at least in the case of children .
hamburger and kaplan have already suggested it in 1996 and we also demonstrated it in a previous work that , instead of an immediate surgery , a regular us follow - up of follicular tumors seems to be a safe and adequate way to decrease unnecessary surgery in adult patients with a fnac diagnosis of possible follicular tumor without atypia .
our suggested approach ( i.e. , watchful waiting ) is a proposal and differs from current guidelines and clinical practice [ 39 , 43 ] .
although recent genetic methods are increasingly popular in this field , the use of these techniques in differential diagnosis of follicular tumors may be questioned until the uniform histological interpretation of follicular lesions is not ensured in our opinion .
the diagnosis of thyroid nodule in hashimoto 's thyroiditis is one of the greatest challenges in thyroidology .
differences in terminology may explain the great differences in occurrence : from 5% in this study to 31.5% [ 43 , 44 ] .
we have found discrete lesions of at least 5 mm in maximal diameter in hashimoto 's thyroiditis in 37.3% of children and only 8.1% of these lesions meet the criteria of a nodule in a pathological sense .
many authors define a thyroid nodule as a discrete lesion within the thyroid gland that is sonographically distinguishable from the adjacent parenchyma [ 39 , 4547 ] .
we think that this definition of nodule in a radiological sense does not correspond to the definition of a nodule in a pathological sense and may lead to an unacceptable high rate of overdiagnosis of discrete lesions in hashimoto 's thyroiditis as nodular goiter with a not negligible iatrogenic psychological harm .
it is well - known that hashimoto 's thyroiditis may be present in as much as 95% of cases as
although the distinction between the so - called pseudonodules and true nodules is not possible in every case [ 50 , 51 ] , our results demonstrate that the correct differential diagnostic of the discrete echo abnormalities is possible in more than 90% of cases by thorough analysis of the echo pattern , the clinical and immunological data .
the significantly higher proportion of adenomas explains the overall worse specificity , positive predictive value , and diagnostic accuracy in children compared with adults because the diagnostic accuracy of fnac is significantly worse for well - known reasons in adenomas than in other lesions .
we categorized those cases differently where the fnac was not diagnostic in the event of spongiform and central type cysts . in accordance with other researchers [ 25 , 26 ]
we have found that the risk of malignancy in these cysts is very low in adults . our practice shows less than 1% ( in this series 0% ) ; therefore , we gave a common us - fnac diagnosis of cystic nodule with less than 1% risk of malignancy in adults , when the cyst was spongiform or it presented central cystic degeneration and lacked abnormal vascularization and microcalcifications .
the significantly greater proportion of nondiagnostic punctures among children may be explained by this difference in categorization .
nevertheless , other authors published even greater rates ( 28% ) of nondiagnostic fnac among children . despite the published data on pediatric fnac , the sensitivity varied between 40% and 100% ; surprisingly great proportion of authors , similarly to us , found no false negative fnac among children [ 10 , 20 , 29 , 5255 ] .
this may be explained by the rare occurrence of papillary microcarcinomas among young patients and the predominance of clinically more aggressive carcinomas among children which can be more easily recognized by fnac . a lower specificity and therefore a lower positive predictive value were found among children compared either to most recent publications or to our adults in our study .
the difference is explained by the occurrence of adenomas which was higher in the former group than in the latter groups . to summarize , childhood nodules are more frequently malignant and carcinomas have a clinically more aggressive clinical presentation in our iodine - deficient area , similarly to published data from iodine - sufficient regions and compared with adults .
our findings of significantly higher rate of adenomas to hyperplastic nodules and the low rate ( in our series the lack of it ) of follicular carcinomas among children require verification by other researchers .
nevertheless , a conservative management of certain types of follicular tumors has to be considered .
spongiform and central type cysts are very rarely malignant in children similarly to adults . if the cytological material is not sufficient to cytodiagnosis in such cysts , we prefer a combined us - fnac diagnosis of cystic nodule with less than 1% risk of malignancy instead of a nondiagnostic cytological report ; this may lessen unnecessary anxiety caused by a nondiagnostic report . | the clinical and pathological presentation of thyroid nodules among younger and adult patients was compared in an iodine - deficient ( i d ) region .
data of 3,010 consecutive patients younger than 20 years and 3,010 patients older than 20 years were compared . the proportion of nodular goiters ( 22.8% versus 39.3% ) , the ratio of surgically treated nodules ( 33.2% versus 15.2% ) , and the proportion of malignant nodules ( 4.3% versus 2.1% ) among diseased patients differed significantly between the two groups ( younger versus adult ) .
nine papillary and 1 medullary carcinoma were found among children , while 15 papillary , 2 follicular , 1 insular , 1 anaplastic , and 1 medullary carcinomas occurred among adults .
the ratio of follicular adenoma to hyperplastic nodules ( 3 : 1 to 1 : 1.67 ) , the proportion of follicular variant ( 77.8% versus 26.7% ) , t4 tumors ( 77.8% versus 33.3% ) , and tumors with lymph node metastasis ( 88.9% versus 66.7% ) were significantly higher among younger papillary carcinoma patients .
no malignancies occurred among spongiform and central type cysts . similarly to iodine - sufficient regions , more nodules are malignant and carcinomas have a clinically more aggressive presentation in children in comparison with adult patients in i d .
taking the significantly greater proportion of adenomas and the lack of follicular carcinoma into account , a conservative approach has to be considered in follicular tumors among children . |
pleomorphic adenoma is a frequent benign neoplasm in the head and neck region and accounts for 70% of all salivary gland tumors .
65% of the tumors arise in the parotid gland , and only 35% involve the minor glands .
its origin in the maxillary antrum is an extremely rare phenomenon as , involvement of the upper respiratory tract by it occurs almost exclusively in the nasal cavity .
the present article reports a unique case of antral pleomorphic adenoma with an extensive ossification accounting to be the second reported case .
a 33-year - old male patient reported in our department with a swelling in the left upper jaw for a period of 9 months . the patient complained a sense of heaviness and occasional pain in the affected area .
he was referred to us from a primary health care center to evaluate any possible odontogenic association to the swelling .
extraoral examination of the patient revealed a diffuse swelling in the left middle third of the face , lateral to the nose with normal skin covering .
it caused obliteration of the left nasolabial fold and slight rising of the left ala of nose .
intraoral observation showed smooth buccal and mild palatal cortical plate expansion in the left maxilla with no surface ulceration [ figure 1 ] .
intraoral photograph of the patient showing buccal and mild palatal cortical plate expansion of the left maxillary bone computerized tomography scan of the face and the maxilla revealed the presence of a huge expansile mass in the left maxillary sinus measuring 47 mm 43 mm that attenuates the antral walls with its expansion in the nasal cavity also [ figure 2 ] . with a provisional diagnosis of odontogenic neoplasm
axial computerized tomography scan demonstrating the huge mass in the left maxillary sinus histological evaluation revealed the presence of the tumor mass composed of overwhelming osteoid seams intermixed with small ducts , nests and strands like structures and some myxoid areas [ figure 3 ] . with the diagnosis of ossifying pleomorphic adenoma hemimaxillectomy
multiple h and e stained sections of the resected specimen showed features consistent with that of the incisional biopsy , justifying our primary diagnosis .
photomicrograph of h and e stained section ( 40 ) showing the tumor mass with an extensive ossification abutting the peripheral fibrous capsule
the term pleomorphic adenoma was first suggested by wills , which designates the benign mixed tumor of the salivary gland , characterized by a combination of epithelioid and connective tissue - like growth .
it is the most frequently encountered salivary gland tumor with the parotid being the most common site .
neoplasms of the minor salivary gland may occur at any location though the palate , upper lip , and buccal mucosa are the three most common sites comprising 82.2% of all minor salivary gland tumors .
pleomorphic adenomas of the upper respiratory tract have been rarely described in the literature and occurs almost exclusively in the nasal cavity with the nasal septum involving 82.590% of the reported incidences .
involvement of the seromucous glands of the maxillary antrum by this tumor is even more rare with minimum reported cases . in a review of 2807 cases of salivary gland neoplasms by spiro ,
ishikawa et al . described intranasal mixed tumors to have no primary involvement of the paranasal sinuses validating its rarity .
facon et al . in the year 2002 reported a unique case of a pleomorphic adenoma of the nasal cavity arising and extending from the medial wall of the maxillary antrum .
the most common clinical presentations include nasal obstruction , discharge , epistaxis , or rhinorrhea .
radiological findings are nonspecific , generally presenting with a well - defined mass with features of bony remodeling rather than bone invasion .
histologically , the pleomorphic adenomas are limited by the outer connective tissue capsule which helps to distinguish the tumor from the surrounding normal tissue .
the tumor comprises diverse patterns with three basic structures : tubuloductal , solid and myxoid areas .
while the ducts are made up of cuboidal cells , spindle and myoepithelial cells comprise the solid areas .
the tumor may show sheets and strands of cells in a mucoid or chondromyxoid background .
the pleomorphic adenomas of the minor salivary glands of aerodigestive tract demonstrate a high cellularity with paucity of stroma contrasting the histological findings of the major salivary gland tumors .
careful monitoring of multiple sections must be done to differentiate it from squamous cell carcinoma , the most common sinonasal malignancy , affecting the maxillary sinus most frequently .
it should be carefully excluded as it most commonly affects the maxillary sinus , accounting 47% to arise in it .
farman et al . reported a rare case of carcinoma ex pleomorphic adenoma of the palate and maxillary sinus with features of adenoid cystic carcinoma and adenocarcinoma in the year 1985 . in our case
though marked myoepithelial cell proliferation was noted in some sections of the excisional sample , absence of cellular pleomorphism , mitotic activity , perineural invasion or squamous metaplasia , ruled out the possibility of the malignant counterpart [ figure 4 ] .
the numerous mature osteoid islands abutting the intact peripheral fibrous capsule with intermixed myxoid areas further suggested that of a benign entity [ figure 5 ] .
the preponderance of osteoid tissue may produce confusion with fibro - osseous lesions arising in the maxilla .
myxoid areas were inconspicuous as ill - defined fine basophilic regions produced by glycosaminoglycan - rich matrix .
though the extensive ossification dominated the histological picture , the connective tissue stroma also showed extreme hyalinization producing cribriform - like patterns .
photomicrograph of h and e stained section ( 400 ) showing cellular elements in a chondromyxoid stroma photomicrograph of h and e stained section ( 100 ) showing glandular elements intermixed with osteoid islands and myxoid areas considering the treatment part , complete surgical resection with histological clear margins produces satisfactory outcomes . | pleomorphic adenoma is the most common benign neoplasm of salivary gland origin involving both major and minor glands .
though parotid is the most common site of origin , it has been reported to arise from various unusual locations .
incidence of its origin in the respiratory tract is extremely rare , and the occurrence is even lower in the maxillary sinus .
we report a case of a huge antral pleomorphic adenoma in a 33-year - old male patient presented as a swelling in the maxilla which was mistaken of odontogenic origin .
histological findings showed extensive ossification and hyalinization of the stroma as a striking feature .
the patient has undergone hemimaxillectomy and is in 8 months follow - up without any sign and symptom of recurrence . |
type 2 diabetes is a major independent risk factor for cvd , and this increased risk is partly attributed to abnormalities in lipid and lipoprotein metabolism as a consequence of hyperglycaemia and insulin resistance [ 14 ] .
people with diabetes have a greater than twofold increase in the risk of cvd , and this risk is even greater in women who have an estimated fivefold higher cardiovascular mortality compared to women without diabetes . additionally , this risk is higher after menopause [ 7 , 8 ] , and with an ageing population the burden of diabetes is likely to increase .
the key lipid abnormalities associated with diabetes include raised ldl - c , vldl - c , and tag concentrations and decreased concentrations of hdl - c [ 1 , 3 , 4 , 9 ] .
dietary intervention plays a major role in the treatment of both type 2 diabetes and dyslipidaemia with particular emphasis on the fatty acid composition of the diet as a determinant of cvd risk [ 9 , 10 ] .
nuts and seeds are rich sources of cis - unsaturated fatty acids , and their inclusion into the diets of people with diabetes is recommended in dietary guidelines [ 11 , 12 ] .
there is strong evidence from both epidemiological and intervention studies for the benefits of incorporating nuts into the diet for cvd prevention and treatment of raised total and ldl cholesterol [ 1321 ] .
these studies have primarily focused on the general population and persons at high risk of cvd , not specifically due to diabetes .
however one prospective study in women with diabetes reported a significant reduction in ldl - c and a 44% reduction in the risk of developing cvd in women consuming 5 or more serves of nuts per week compared to those who almost never consume nuts .
only a small number of intervention studies have examined the inclusion of nuts in the diets of persons with type 2 diabetes , and these have produced inconsistent results [ 2327 ] . while two studies have reported reductions in ldl - c when compared to control diets [ 23 , 27 ] , others have only shown reductions compared to baseline with no difference when compared to control diets [ 2426 ] .
blood triglyceride concentrations have been relatively unaffected by the inclusion of nuts in the diet of those with diabetes [ 2327 ] .
limitations with these studies include relatively low initial concentrations of ldl - c [ 24 , 26 , 27 ] , high dropout rates , or weight loss which may have confounded the results . therefore to date there is limited evidence for a link between nut consumption and improved blood lipid profiles in people with type 2 diabetes .
although studies in the general population have reported reductions in cardiovascular risk factors with the consumption of seeds [ 28 , 29 ] , to date there have been no intervention studies examining the incorporation of seeds into the diet of people with diabetes .
previous research suggests that pufa - rich diets are associated with greater reductions in ldl - c , triglyceride , and hdl - c compared to monounsaturated - fatty - acid-(mufa- ) enriched diets .
given the predominant fatty acids in most nuts are mufa , it is hypothesised that the consumption of seeds might influence different cardiovascular outcomes in comparison to nuts .
these differences may be important in addressing a number of key lipid abnormalities associated with type 2 diabetes . to investigate this we supplied personalised diets based on the new zealand and european guidelines supplemented with either almonds or sunflower seeds to postmenopausal women with type 2 diabetes , to assess the effects on cvd - mediated risk factors and glycaemic control .
twenty - two postmenopausal women ( mean age 62 5.7 years ) with type 2 diabetes were recruited into the study from general practitioners ( gps ) practices in the dunedin area , the diabetes clinic at dunedin hospital , south link health and advertisements in local newspapers .
inclusion criteria were postmenopausal status and type 2 diabetes mellitus as diagnosed by the women 's medical practitioners according to established criteria .
the participants also needed to be able to report to the metabolic facility in the department of human nutrition , university of otago at midday from monday to friday during the dietary intervention periods .
exclusion criteria were current use or use within the previous year of insulin , lipid - lowering drugs or hormone replacement therapy , present smoking , allergies to nuts or seeds , or a tc above 8 mmol / l .
all participants gave written informed consent , and the study was approved by the otago ethics committee .
this study utilised a randomised crossover design with two three - week interventions ( ad and skd ) separated by a four - week washout when the participants returned to their habitual diets .
two blood samples were collected by venepuncture 48 hours apart after an overnight fast , at the beginning and in the final week of each intervention period .
lipids , lipoproteins , apolipoproteins a and b , oxidised ldl , insulin , vitamin e , glucose , and glycosylated haemoglobin ( hba1c ) were measured .
height was measured at the beginning of the study to the nearest millimetre using a stadiometer .
participants were weighed , to the nearest 0.01 kilogram on calibrated electronic scales ( wedderburn scales , auckland , new zealand ) , at each blood draw and weekly during the intervention periods .
blood pressure was measured after a 5-minute rest at baseline and at the end of each dietary treatment using a random zero sphygmomanometer ( hawksley & sons ltd , lancing , uk ) .
participants were asked to maintain their usual level of physical activity during the course of the study . a four - day weighed diet record ( 4ddr ) and a prestudy interview formed the basis for the personalised diets .
participants were asked to complete a 4ddr before the start of the dietary intervention and during the washout phase prior to the second dietary phase to assess habitual dietary intake .
kent , uk ) accurate to 2 g , a measuring cup , and measuring spoons and given detailed verbal and written instructions on the method of completing an accurate food record .
after completion of the baseline diet record the participants were individually interviewed to clarify any issues raised by the written records and to ascertain their likes and dislikes .
personalised diets to be provided during the dietary intervention were matched to each subject 's habitual portion size intake to assist with the maintenance of a stable weight .
the diet records were analysed using the computer programme diet cruncher for macintosh ( marshall - seeley , version 1.2.0 , 2001 ) , which utilises food composition data from the concise new zealand food composition tables .
the study diet was a recommended dietary pattern for persons with type 2 diabetes based on the european and new zealand evidence - based guidelines [ 11 , 12 ] .
the diets were designed to provide 30% total energy from fat of which 8% was to come from saturated fat , 15% or 9% from monounsaturated fat , and 6% or 12% from polyunsaturated fat in the ad and skd diets , respectively .
the dietary fibre target was 2535 g and dietary cholesterol 200 mg per day .
a three - week menu cycle was developed incorporating the recommended daily dietary pattern with two or more portions of fruit and three or more portions of vegetables , six portions of breads and cereals predominantly wholegrain , two portions of low fat milk and dairy products , and one portion of lean meat , poultry , fish , or eggs .
fish and a vegetarian dish were each incorporated in the main meal once every week .
all meals were prepared with minimal fat , salt , and sugar , and any snacks or preprepared foods used were also low in fat , salt , and sugar .
thirty grams of whole natural almonds or 30 g sunflower kernels were added to the ad or skd diets , respectively , each day of the three - week intervention period . from monday to friday they were consumed as part of the midday meal .
almonds and sunflower kernels were chosen as they have a similar amount of saturated fat per 100 g and similar total amounts of unsaturated fats , almonds being rich in monounsaturated fat and sunflower kernels in polyunsaturated fat .
the small amounts of added fats and oils needed for spreading , baking , or cooking reflected the composition of the almonds and sunflower kernels . a predominantly monounsaturated spread ( olivio , unilever , auckland , new zealand ) and olive oil ( lupi extra virgin , william aitken & co. , auckland , new zealand ) were used in the almond diet and a predominantly polyunsaturated spread ( meadow lea , goodman fielder , auckland , new zealand ) and grape seed oil ( azalea , william aitken & co. , auckland , new zealand ) in the sunflower kernel diet .
thus the nutrient composition of the ad and skd was planned to be essentially the same apart from the differing fatty acid composition .
participants reported to the metabolic facility at midday during the week to consume the dinner meal and take home the remaining meal items for the following 24 hours . on fridays
, participants were supplied with their entire food requirements for the weekend up until midday monday .
beverages were not provided as part of the diet distributed by the metabolic facility but the participants kept a daily record of what they drank each day .
however , a list of suitable snack foods was given to each participant to choose from if required , and they were instructed to weigh and record these foods .
electronic scales , measuring cups , and measuring spoons were given to each participant to keep throughout the dietary intervention to assist with the measurement of beverages or extra foods consumed .
furthermore , participants were asked to weigh and record any food from the metabolic facility that was not consumed . during the washout period
the participants were counseled to return to the diet , which they had consumed prior to the study , and to avoid eating nuts and nut products or seeds and seed products .
blood samples were collected into vacutainers ( belton dickinson diagnostics ) containing dipotassium edta ( for analysis of lipids , lipoprotein , and -tocopherol ) , sodium fluoride ( for analysis of hba1c and glucose ) , and tubes containing no additives ( for analysis of insulin ) .
the duplicate blood tests were drawn approximately 48 hours apart to account for intraindividual variation in blood cholesterol concentration .
once plasma / serum and red blood cells were separated , aliquots were stored at 80c until laboratory analyses were conducted . plasma tc
, hdl - c , tag , glucose , and hba1c concentrations were measured by enzymatic methods using kits and calibrators supplied by roche diagnostics ( mannheim , germany ) on a cobas mira plus analyser .
hdl - c was measured in the supernatant following precipitation of apoprotein b containing lipoproteins with phosphotungstate - magnesium chloride solution .
coefficient of variation ( cv ) for measurement of tc was 1.6% , hdl - c 2.8% , tag 4.5% , glucose 1.8% , and hba1c 1.5% .
apolipoproteins a1 and b100 and -tocopherol concentrations were measured on one of the two nonconsecutive blood samples during each period .
apolipoprotein a1 and b100 concentrations were determined by immunoturbidimetry using commercial kits from roche diagnostics ( mannheim , germany ) .
plasma -tocopherol was determined using the agilent high - performance liquid chromatography system ( 1100 series ) based on methods described by thurnham et al . .
serum insulin was measured by a solid - phase radioimmunoassay using coat - a - count kits from diagnostic products corporation ( los angeles , united states ) .
plasma oxidised low - density lipoprotein ( oxldl ) was determined by using the oxidised ldl enzyme - linked immunosorbent assay ( elisa ) kit from mercodia ( uppsala , sweden ) .
calibration and quality control were maintained by participation in the royal australasian college of pathologists association quality assurance program .
a sample size of 22 women was calculated to have an 80% chance of detecting a difference of 0.36 mmol / l in ldl cholesterol at the 5% level of significance .
the study was primarily set up to investigate the difference in change in variables between the two diet treatments .
this was analysed using a mixed model , which included a term for period and takes into account the underlying correlation between the repeated measures .
these results were adjusted for baseline and order effects . the variance in change between the diets
is presented as mean and 95% confidence intervals . due to glucose , insulin , vitamin e , and oxidised ldl data being skewed ,
the results required logarithmic transformation . thus the results are presented as the ratio of the geometric means with 95% confidence intervals .
further analyses were conducted to investigate the effect of each diet by using a paired t - test to analyse the difference between baseline and final values .
statistical analyses were performed on dietary data using computer software spss 11.0.2 ( spss for mac os x , 2003 , spss incorporated ) .
a total of 22 women were recruited and completed both the almond and sunflower kernel dietary interventions .
the data from the 4ddr completed by each participant before each dietary intervention was pooled .
furthermore , a diet record was completed for each participant , each day of both diet interventions , and data pooled for the ad and skd and presented as mean values .
there were no significant differences in total energy , protein , carbohydrate , and cholesterol levels between the diets ( table 1 ) .
when the ad was compared to the skd , there was no significant difference in the total fat content which provided 30.3% and 30.4% total energy from fat sources , respectively .
both ad and skd diets contributed a similar amount of safa 8.0% and 7.8% , respectively ; however as intended the ad had almost twice the amount of mufa compared to the skd , 15% versus 8.9% ( p < 0.001 ) .
the reverse was true with respect to pufa with the skd providing 12.5% total energy compared to only 6.3% total energy in the ad ( p < 0.001 ) , which was the desired target .
fibre and vitamin e intakes were significantly different between the diets ( 0.028 , p < 0.001 , resp . ) .
when compared to the habitual diet , the ad had significant reductions in energy , total fat , safa and cholesterol ( all p < 0.001 ) , and protein ( p < 0.01 ) .
fibre and vitamin e intakes were higher in ad compared to baseline ( both p < 0.001 ) . on the skd there were significant reductions in energy , protein , and total fat ( all p < 0.01 ) and safa , mufa , and cholesterol ( all p < 0.001 ) intakes when compared to the habitual diet . in comparison there were significant increases in pufa and vitamin e ( p < 0.001 ) , fibre ( p < 0.01 ) , and carbohydrate ( p < 0.05 ) intakes .
nutrient contribution of the whole almonds and sunflower kernels to the respective diets was very similar .
one serve of either whole almonds ( 30 grams ) or sunflower kernels ( 30 grams ) per day contributed 11% of total daily energy and provided 15.7 grams and 14.9 grams of fat per day , respectively , which equated to approximately one - third of the total fat content of the diet .
there were significant differences between the dietary treatments for hdl - c , tag , apo a1 , and apo b100 .
the ad diet essentially maintained baseline hdl - c concentrations with a drop of 0.01 mmol / l , whereas there was a significant 0.07 mmol / l reduction ( p = 0.050 ) observed on the skd .
a 12% reduction in tag concentrations was observed on the skd ( p = 0.001 ) , whereas no significant reduction was seen on the ad ( p = 0.798 ) .
the skd reduced apo a1 concentrations to a greater extent than the ad diet ( p = 0.001 ) .
similar to apo a1 results , the reduction in apo b100 concentrations was also significantly greater on skd compared to ad ( p < 0.001 ) .
there were significant reductions in tc ( 5.6% ad and 9% skd ) , ldl - c ( 7.4% ad and 9.5% skd ) , apo a1 ( 2.8% ad and 6.9% skd ) , apob ( 4.8% ad and 10% skd ) , and oxldl ( 7% ad and 12% skd ) on both dietary treatments ( table 2 ) .
overall there were no differences in the change of sbp ( p = 0.630 ) and dbp ( p = 0.719 ) between the two dietary treatments ( table 2 ) .
however there was a drop in both sbp and dbp levels from baseline to the end of the two dietary treatments .
the decrease in dbp from baseline was significant on both diets ad ( 4.6% , p = 0.020 ) and skd ( 3.4% , p = 0.004 ) .
however the drop in sbp from baseline was only significant on the skd ( 5.0% , p = 0.016 ) .
there was no significant difference in changes in blood glucose concentrations between the two dietary treatments ( table 3 ) .
however 10% ( p = 0.006 ) and 11% ( p = 0.002 ) reductions in blood glucose concentrations from baseline to end of treatment were observed on the ad and skd , respectively .
a significant reduction in hba1c was also found on both diets ( 2.6% , p = 0.020 for ad and 3.3% p = 0.017 for skd ) with no significant difference between dietary treatments ( p = 0.091 ) . there were no differences in the change in insulin observed on the two dietary treatments .
however when compared to baseline , a significant reduction ( p = 0.010 ) was observed on the skd diet .
the skd caused a reduction in plasma -tocopherol concentrations ( 4.4% ) , whereas the ad diet increased -tocopherol ( 3.4% ) ( table 2 ) .
there was no significant difference in change in body weight between the two dietary treatments ( table 4 ) .
however a significant reduction in weight was observed when change from the start to end of each dietary phase was compared ( p < 0.001 for ad and skd ) .
the mean bmi results at the start of the ad and skd were 29.16 and 29.24 kg / m , respectively .
there was a reduction in bmi seen on both diets by 0.33 kg / m ( ad , p < 0.001 ) and 0.32 kg / m
( skd , p < 0.001 ) ; however there was no difference between dietary treatments ( p = 0.587 ) .
as far as we are aware this is the first study to compare the effects of almonds and sunflower kernels in people with type 2 diabetes , and it reinforces the premise that dietary intervention plays a major role in the treatment of both type 2 diabetes and dyslipidaemia .
the study demonstrated clinically important improvements in cvd risk factors and glycaemic control in postmenopausal women with type 2 diabetes , who were provided with personalised diets with the addition of either almonds or sunflower kernels . in spite of this
however the cost of providing all the food for the two intervention periods of three weeks each and the considerable participant burden involved in taking part curtailed any increase in participant numbers or time .
in addition the study was powered on ldl cholesterol , and the number of women and the intervention period were both calculated to be adequate to see changes in the lipid and lipoprotein end points of interest . in order to see definitive changes in hba1c an intervention period of at least eight weeks would have been necessary .
both diets resulted in beneficial effects on cardiovascular risk factors , namely , a reduction in tc , ldl - c , and oxidised ldl .
the hdl - c concentrations were maintained on the ad , but fell on the skd diet .
previous studies have reported reductions or increases in hdl - c with the addition of nuts to the diets of people with diabetes .
our study suggests that the inclusion of nuts may be beneficial in maintaining hdl - c concentrations , and it is likely that the high monounsaturated content of the almonds contributes to this effect .
a meta - analysis by mensink and katan on fat type suggested some differences between mufa and pufa , favouring mufa for beneficial effects on hdl - c .
the skd diet was predominantly pufa , suggesting the difference in fatty acids is most likely to account for the observed difference .
hodson et al . observed a 14% reduction of hdl on a diet containing 9.1% pufa and a 4% reduction in hdl with a predominantly mufa ( 11.6% ) diet .
the importance of hdl as a cvd protective factor at all ages and especially in women has recently been highlighted in the analysis of the score dataset . a further difference between dietary treatments was a reduction in tag on the skd compared to the ad diet .
studies in the general population have reported beneficial reductions in tag concentrations on a pufa - rich diet . however , as with our study , most other studies in people with diabetes , which have included regular nut consumption , have shown no changes in tag concentrations [ 2327 ] . given that low hdl - c and high tag
are often observed in people with diabetes [ 1 , 3 , 4 , 9 ] , the use of both nuts and seeds would appear to offer a sensible approach to improve these metabolic parameters .
however the varieties , combinations , and amounts would need to be tested in a further intervention .
the reductions in ldl - c observed in our study are similar to other studies where nuts have been included in the diet of people with diabetes [ 23 , 25 , 27 ] .
the significant reduction in ldl - c is likely to be due to the favourable cis - unsaturated fat content of both diets .
mensink and katan reported a slight beneficial effect of n-6 pufa on reducing concentrations of ldl - c . in the present study although the reduction in ldl - c was not significantly different between the ad and skd diets , the magnitude of the reduction was higher for the polyunsaturated - rich skd .
the cholesterol lowering properties of seeds were also demonstrated by wu and colleagues who conducted a randomized , placebo - controlled , crossover study of two intervention periods separated by a three - week washout with 26 healthy postmenopausal women .
participants consumed either 50 g / d sesame seed or the placebo of 50 g / d rice powder . following the sesame intervention plasma total cholesterol ( tc ) , ldl - c , and the ratio of ldl - c to hdl - c decreased by 5 , 10 , and 6% , respectively , which was significantly different from the placebo .
both the ad and skd diets were equally effective at controlling glycaemic response , with reductions in hba1c , blood glucose , and insulin .
a recent trial , powered to show reductions in hba1c , and which used intensive individualised dietary advice given to optimally medicated individuals with type 2 diabetes , reported reductions of 0.4% between intervention and usual dietary care .
there is a continuous relationship between complications of diabetes and hba1c suggesting that the modest reductions observed in our study could be clinically important .
2009 , who reported a 20% reduction with the consumption of 30 g of walnuts per day .
2010 , reported only a 4.1% reduction in fasting insulin concentrations with the addition of 60 g of almonds .
almonds , sunflower seeds , and walnuts are all rich sources of cis - unsaturated fat .
the addition of modest quantities of these foods appears to improve glycaemic control , with the pufa - rich foods having a more pronounced effect .
both nuts and seeds are low in sodium and contain a variety of constituents including unsaturated fatty acids , magnesium , potassium , and other bioactive compounds , which may have beneficial effects on blood pressure .
few studies have investigated the effects of nuts on blood pressure . in the physicians ' health study
those who consumed nuts on a daily basis had a significantly reduced risk of hypertension compared to those who did not consume nuts .
2006 , reported a reduction in both systolic and diastolic blood pressure in response to a mediterranean - style diet containing 30 g / day nuts compared to a low fat control .
this suggests that maybe the cis - unsaturated fat content of the diet may be playing an important role in bp reduction . in our study
both nuts ( rich in mufa ) and seeds ( rich in pufa ) appeared equally effective at reducing bp .
converting recommended dietary fatty acid percentages into practical food - based advice in order to reach lipid targets is a challenging part of nutrition counselling .
general advice to increase unsaturated fat may inadvertently result in increased saturated fat and energy intakes .
a practical approach where foods high in unsaturated fat are added to a recommended diet may be simpler and enhance compliance to dietary change .
nuts , although high in energy , have demonstrated strong dietary compensation with little change in body weight when consumed in the long term [ 4245 ] .
therefore the addition of nuts to an optimal diet may be an appropriate approach for reaching dietary fat targets .
the participants were highly motivated to adhere to their diets , and as none of them were on lipid lowering medication , this intervention gives an indication of the potential effect on lipids of diet alone . in terms of acceptability
a recent study has demonstrated that the recommended amount of 30 g / d of hazelnuts , consumed over 12 weeks is both achievable and sustainable .
the addition of nuts and seeds into the diets of people with diabetes is a simple intervention that can be effective in helping to achieve the lipid targets recommended .
given the increasing rates of diabetes and the greater risk of cvd in postmenopausal women who have diabetes , it is timely to develop easily implemented dietary strategies to reduce the risk factors associated with cardiovascular disease .
this study suggests that the inclusion of either nuts and seeds into a diet based on current evidence - based recommendations is beneficial and that this benefit was seen in women who were not on lipid lowering medication .
given the differential effects of nuts and seeds on hdl - c and tag , it seems prudent to advise the inclusion of both in the diets of people with diabetes .
a trial over a longer time with a larger group in a community setting , testing a combination of almonds and sunflower seeds , could be of interest . | dietary guidelines for the treatment of type 2 diabetes advocate the regular consumption of nuts and seeds .
key lipid abnormalities associated with diabetes include raised ldl - c , vldl - c , and tag concentrations and decreased concentrations of hdl - c .
the fatty acid profiles of nuts and seeds differ and may potentially influence lipid outcomes in people with diabetes differently . to examine the effects of nut or seed consumption on lipid and lipoprotein markers of cardiovascular disease ( cvd ) , we added almonds ( ad ) or sunflower kernels ( skd ) to a recommended diet in a randomised crossover feeding study .
twenty - two postmenopausal women with type 2 diabetes consumed personalised diets , with the addition of 30 g / d of either almonds or sunflower kernels .
all food was supplied for two periods of three weeks , separated by a four - week washout .
there was a significant reduction in high - density lipoprotein cholesterol ( hdl - c ) , triacylglycerol ( tag ) , and apolipoprotein ( apo ) a1 and b100 on the skd compared to the ad .
total ( tc ) and low density lipoprotein cholesterol ( ldl - c ) decreased significantly on both diets from baseline , with no difference between diets . a diet with the addition of either almonds or sunflower kernels has clinically beneficial effects on lipid- and lipoprotein - mediated cvd risk . |
pancreatic cancer is a major cause of cancer - related mortality worldwide as it ranks as the 5th leading cause of death , with an annual incidence in certain countries of approximately 20,00040,000 cases and poor mortality rates .
owning to the high frequency of local extension and/or metastatic disease at the time of diagnosis , only a small minority of patients are candidates for curative resection . moreover ,
surgery alone is limited , with an unsatisfactory prognosis and a high incidence of postoperative recurrence . to improve the survival of patients with pancreatic cancer ,
effective multimodality treatments are demonstrating effective disease control and prolongation of quality of life time .
a recent randomized controlled study demonstrated that treatment with gemcitabine exhibited a better clinical benefit response ( cbr ) ( 23.8 versus 4.8% ) and median survival period ( 5.65 versus 4.41 months ) than bolus 5-fluorouracil ( 5-fu ) .
however , current chemotherapy regimens for pancreatic cancer must continue to be improved since gemcitabine alone offers limited survival benefit .
gemcitabine administration with a fixed dose - rate infusion and gemcitabine combination regimens have been investigated , but a meaningful impact on survival , compared with that of gemcitabine monotherapy , has not been reached .
recent randomized phase iii studies of gemcitabine plus erlotinib and gemcitabine plus capecitabine have demonstrated significant survival benefit , but a consensus on the optimal first line therapy has not been agreed upon .
liver dominant and liver refractory metastatic diseases still remain common problems in the management of metastatic pancreatic cancer .
transcatheter arterial embolization ( tae ) and transcatheter arterial chemoembolization ( tace ) are increasingly used as regional therapeutic modalities for the treatment of unresectable hepatic malignancies . in general , tae and tace
have been used when surgical resection and/or systemic therapy have failed to produce an adequate response or when conventional therapy has been known to be ineffective .
there has been almost no information reported on the use of tace in hepatic metastases arising from pancreatic cancer .
drug - eluting bead tace is a drug delivery system that combines the local embolization of vasculature with the release of chemotherapy into adjacent tissue .
its administration is similar to that of conventional tace , and it represents a minimally invasive procedure performed by interventional radiologists [ 8 , 9 ] .
early phase 1 and nonrandomized phase 2 studies have confirmed the ability of this device to deliver a local , controlled , sustained dose of doxorubicin to the tumors , with minimal systemic doxorubicin exposure .
a recently completed randomized phase 2 study demonstrated that these drug - eluting doxorubicin beads had improved response rates when compared to conventional tace in advanced hcc and significantly less overall adverse events , including doxorubicin - related side effects .
therefore , we conducted a prospective observational study to evaluate the safety and efficacy of hepatic arterial therapy in the management of liver dominant or liver refractory metastatic pancreatic cancer .
an irb - approved prospective multi - institutional treatment registry of 805 patients undergoing 1358 treatments for primary or secondary cancers in the liver was evaluated from january 2007 to january 2011 .
of the 805 patients , 10 patients presented with liver dominant metastatic pancreatic cancer to the liver and were treated with doxorubicin or irinotecan drug - eluting beads .
the registry was designed to satisfy the strict criteria for critical appraising of the quality of a registry study with ( 1 ) a well - described patient population , ( 2 ) hypothesis generating and answering questions , ( 3 ) high quality data , with good quality control , ( 4 ) independent assessment of outcomes , ( 5 ) good clinically relevant followup with minimal loss of patients , and ( 6 ) comparable patient evaluation across all institutional participating .
patients were included for therapy if they were 18 years of age or older , of any race or sex , had histologic and radiologic proof of metastatic pancreatic cancer to the liver by percutaneous biopsy , were able to give informed consent , and were eligible for treatment as previously described [ 13 , 14 ] .
patients must have had an ecog performance status score of less than or equal to 2 with a life expectancy of greater than or equal to 3 months , nonpregnant with an acceptable contraceptive in premenopausal women .
exclusion to therapy was contraindication to angiographic and selective visceral catheterization , significant extrahepatic disease , representing an eminent life - threatening outcome , greater than 75% of hepatic parenchymal involvement , severe liver dysfunction , pregnancy , and severe cardiac comorbidities . only patients with liver dominant ( defined as greater than 50% of the overall total disease burden ) were considered for treatment .
patients were followed for any treatment - related adverse experiences for 30 days after each treatment and monitored for survival for two years .
follow - up assessments included a triphase ct scan of the liver within at least one to two months from the treatment completion with the evaluation of the enhancement pattern of the target lesion and tumor response rates measured according to modified recist criteria . the decision to treat with deb
was based on either the need to dose reduce chemotherapy based on toxicity and maintain disease control or because of hepatic specific progression .
given that it is well established that pancreatic adenocarcinoma is a systemic disease , the use of concurrent chemotherapy and deb was performed in all patients .
the use of deb in neuroendocrine ( net ) tumors was based on the established disease biology and the extent of liver involvement .
defining the amount of liver disease was integral to defining both the number of treatments and the type of catheter position and therapy that would be performed . for finite numbers of lesions , defined as less than four lesions ,
a treatment cycle was planned for a minimum of two dosing schedules of at least 100 mg of debdox to 150 mg of debdox [ 16 , 17 ] loaded in two bead vials or 100 mg debiri loaded in one vial .
if toxicity and performance status changes during treatment , the intervals between deb are extended .
repeat abdominal imaging every three months from the initial first treatment cycle is recommended to evaluate response as well as planned retreatment .
a treatment is hepatic arterial chemotherapy to one single lobe , which could also be a treatment cycle if a patient has only unilobar disease . for diffuse disease , a plan of a minimum of four doses of 100 to 150 mg ( depending on the extent of tumor burden and the extent of hepatic parenchyma reserve )
is loaded into one or two bead vials of the similar size as above with the plan for at least two treatments per lobe with every three to four week dosing schedule , following toxicity , and extending the interval if toxicity was seen with planned repeat ct scan three months from the first dose to evaluate tumor response .
for example , if patients present with bilobar disease , they would receive the first bead treatment to the right lobe , then three weeks after the second bead treatment to the left lobe , then three weeks later third bead treatment to the right lobe , and then again three weeks later to the left lobe .
the reason for lobar infusion is based on the desire for drug delivery and less on inducing stasis in patients with multifocal lobar disease that is not amenable to superselective delivery .
additional embolic material is not recommended since prior studies have demonstrated the increase in adverse events but no benefit in response rates .
all bead therapies were performed with the dc / lc bead microspheres ( drug - eluting bead ( deb ) ; http://www.biocompatibles.com/ , biocompatibles uk , surrey , uk ) .
ten patients underwent a total of 17 treatment sessions with drug - eluting beads ( debs ) ( table 1 ) .
there were 6 females and 4 males with a median age of 69 years and a range of 45 to 77 years .
2 had a history of tobacco use with a median 40 pack per year history .
all 10 patients had a primary pancreatic tumor ; 6 ( 60% ) with an adenocarcinoma or 4 ( 40% ) with a neuroendocrine tumor ( net ) .
six patients had a distinct number of hepatic lesions , and 4 patients had lesions which could not be quantified .
seven ( 70% ) patients had < 25% liver involvement , while 3 ( 30% ) had 2650% liver involvement . of the 10 patients , 2 ( 20% ) had undergone a prior liver resection ( table 2 ) .
seven ( 70% ) patients ( 6 with adeno and 1 with net ) had had prior chemotherapy ; 4 with gemcitabine and 1 each with folfox , folfiri , and doxorubicin / streptozocin . in these 6 patients with adenocarcinoma ,
5 had grade 3 - 4 either hematologic or neurologic toxicity that led to either a dose - delayed , dose - limiting toxicity or discontinuation of one of the chemotherapeutic agents .
four patients had local liver lesion disease progression with either new lesions or the growth of established lesions while on chemotherapy .
three patients had had prior trans - arterial chemoembolization ( tace ) ; 2 with cam and 1 with carboplatin , with only stable disease as best response and the reason for deb therapy . finally , 6 ( 60% ) patients were undergoing concurrent chemotherapy , either to control small volume extrahepatic disease or based on the desire of the treating multidisciplinary team in order to obtain synergistic results with systemic chemotherapy and deb therapy .
there were 17 total treatments , with a median of 1.5 bead courses per patient , ranging from 1 to 3 courses per patient ( table 3 ) .
pancreatic adenocarcinoma patients were all treated with irinotecan beads , and net patient were treated with doxorubicin beads .
the total hepatic dose exposure for all the irinotecan beads was 250 , with a range of 100 to 400 , and 150 for the doxorubicin beads with a range of 100 to 450 . for 13 ( 76% ) of the treatments
, we utilized 100 to 300 micron beads , and , for the remaining 4 ( 24% ) treatments , we chose to use 300 to 500 micron beads .
lab values showed an average decrease of 0.23 cells/l in white blood cells ( range of 0.453.00 cells / ul ) . on average ,
a patient 's hemoglobin increased by 0.8 g / dl ( range 1.12.1 g / dl ) . in a complete evaluation of all hematologic parameters
we found only minimal changes in overall hematologic values as long as established bead size , technique , and dosages were utilized .
a total of 7 adverse events consisting of vomiting , pain , nausea , and gastritis were recorded ( table 4 ) .
none of these adverse events were grade 3 or higher , which was the cutoff for being considered a severe adverse event .
the vomiting , pain , and nausea were consistent with postembolic syndrome and were found to be related more commonly to debiri administration in the adenocarcinoma patients .
the patients were set to be followed at 3 , 6 , 9 , 12 , and 18 months .
their response rates were measured using a modified response evaluation criteria in solid tumors ( mrecist ) criteria ( table 5 ) .
no patients died due to complications of the procedure within 90 days of the treatment . at 3 months ( n = 10 ) , 4 ( 40% ) patients had a complete response to their treatment and 6 ( 60% ) patients had a partial response . at 6 months
, 2 patients had died due to their disease . of the remaining 8 patients , 3 had a complete response . at the 9 month interval ( n
= 8) , 2 ( 25% ) patients had a complete response , 5 ( 63% ) had a partial response , and 1 ( 13% ) had stable disease . at the 12-month followup , 1 more
( n = 7 ) , 2 ( 25% ) patients had stable disease , 2 ( 25% ) had a complete response , and 3 ( 50% ) had a partial response . finally , at the 18 month followup ( n = 6 ) , 1 other patient had died of disease .
another patient 's ( n = 1 ) disease had progressed , 1 had a complete response , and 1 had stable disease .
at the time of this writing , 4 patients had still not reached the time of the followup .
after a median followup of 16 months , the 6- and 12-month response rates was 80% and 75% , respectively , with a median overall survival of 9.3 months .
there remains limited information and limited clinical guidelines on the management of the subset of patients with liver predominant metastasis of pancreatic adenocarcinoma .
however , by using what we know about treating similarly metastasized neuroendocrine tumors and other treatment modalities for adenocarcinoma , we hope to gain some better understanding of how to effectively treat patients with liver predominant metastatic pancreatic adenocarcinoma .
de baere et al . demonstrated the safety and efficacy of debdox with tace to treat hepatic metastases from well - differentiated neuroendocrine tumors . in this study , 20 patients underwent 34 sessions of tace with debs loaded with doxorubicin ( 500700 micron beads ) . at 3 months ,
80 percent of patients had a partial response , 3 patients had stable disease , and 1 had progressive disease .
patients had a median time of 15 months to progression of disease . in our study , no patients had signs of progression up to 12 months of follow - up .
67% of patients had postembolization syndrome which lasted less than 7 days , and 5 patients showed signs of tace - induced peripheral liver necrosis at 1 month .
it was concluded that tace with debs was well tolerated and safe but needs more studies to define the best protocol to use such treatments .
our study used a maximum of 300500 micron size beads as compared to the 500700 micron beads used in the study by de baere et al . while all the patients in their study had metastatic neuroendocrine tumors , 4 out of 10 ( 40% ) of our patients had neuroendocrine tumors , while the remaining 6 ( 60% ) had primary pancreatic adenocarcinoma .
while the use of debdox and debiri in liver predominant metastases from pancreatic carcinoma is one modality to treat this condition , others have proven successful with a different approach using tace . in a recent case report , brown et al .
examines the complete radiographic response in a patient with liver isolated metastases from pancreatic adenocarcinoma . for this particular patient , conventional tace with gemcitabine and cisplatin was used .
this patient had a grade iii poorly differentiated ductal adenocarcinoma that demonstrated hepatic metastasis on a follow - up ct at 14 months after recovery .
recist criteria were also used to evaluate this patient 's response . at a 7-month followup ,
the patient showed no evidence of malignancy , and ct / pet showed no hypermetabolic foci .
gemcitabine has been used systemically in the past for the treatment of pancreatic adenocarcinoma with only marginal activity .
the use of gemcitabine using tace proved more efficacious in the case report by brown et al .
this increased response to liver - directed therapy may suggest a role for tace in the treatment of liver dominant metastases from pancreatic adenocarcinoma . besides tace
okusaka et al . have reported success using s-1 , an oral fluoropyrimidine derivative in patients with metastatic pancreatic adenocarcinoma .
the patients treated in this study had inoperable pancreatic cancer and were administered a predetermined dose of s-1 based on their body surface area . in total ,
0% of patients achieved complete response , 37.5% of patients achieved a partial response , while 32.5% of patients had progressive disease .
the median time to progression was 3.7 months , and the median survival time was 9.2 months .
this study had a larger patient population than our study , but we achieved a comparable median survival time of 9.3 months . a limitation of this study may be that different doses and bead sizes were administered to each patient based on the physician 's discretion . also , our observations were based on a small patient population . while there is much data on the treatment of metastatic neuroendocrine tumors , we hope to shed some light on the efficacy of our liver - directed therapy for metastatic pancreatic adenocarcinoma .
our study was based on a multi - institutional registry showing that the data is generalizable to any institution .
while our patients showed some adverse effects , they were limited to vomiting , pain , and nausea and none were classified as severe
. the limited toxicity profile of this treatment is beneficial for the patient with an already terminal illness .
while the median survival of patients with metastatic pancreatic adenocarcinoma is 36 months , we achieved a median overall survival of 9.3 months .
future studies with reproducible results are needed to establish this treatment modality as the preferred form for metastatic pancreatic adenocarcinoma .
we believe the results of our study show promise for its use in the future . | introduction . there has been limited reporting on the use of hepatic - directed therapy in liver dominant hepatic metastases arising from pancreatic cancer .
methods .
an irb - approved prospective multi - institutional treatment registry of 885 patients undergoing 1458 treatments for primary or secondary cancers in the liver was evaluated from january 2007 to january 2011 .
results .
ten patients underwent a total of 17 treatment sessions with drug - eluting beads ( debs ) .
six patients received concurrent chemotherapy while undergoing deb with no severe adverse events .
after a median followup of 16 months , the 6- and 12-month response rates were 80% and 75% , respectively , with a median overall survival of 9.3 months .
conclusion .
hepatic arterial therapy with deb can be safely and effectively used in selected patients with liver predominant metastatic disease from pancreatic cancer .
this therapy should be considered in combination with systemic chemotherapy as a possible second therapy given the limited response rates of second - line chemotherapy . |
numerous large - scale clinical trials have demonstrated that statins ( 3-hydroxy-3-methylglutaryl - coenzyme a [ hmg - coa ] reductase inhibitors ) substantially reduce cardiovascular ( cv ) morbidity and mortality in both primary and secondary prevention of cv disease ( cvd ) .
a meta - analysis of individual data from 26 randomized trials of statins reported a 10% proportional reduction in all - cause mortality and a 20% proportional reduction in cv death/1.0 mmol / l ( 40 mg / dl ) low - density lipoprotein - cholesterol ( ldl - c ) reduction .
the risk of major coronary events was reduced by 23% , and the risk of stroke was reduced by 17%/mmol / l ( 40 mg / dl ) ldl - c reduction .
thus , most international guidelines on cvd prevention in clinical practice consider statin as the first - line medication of lipid - lowering treatment and recommend modulating the intensity of the preventive intervention according to the level of the total cv risk .
atorvastatin , a member of the statins , was first synthesized in 1985 . since its approval by the us food and drug administration in 1996 ,
atorvastatin has become one of the best - selling drugs in pharmaceutical history . in china
, atorvastatin is the most widely used lipid - lowering medication , accounting for about 40% of total statin use in secondary prevention of cvd . besides
, the efficacy and safety of atorvastatin have been testified by more than 200 randomized controlled trials ( rcts ) , with most sufficient clinical evidence among all statins .
it is , therefore , important to evaluate the effect of atorvastatin in chinese population . in this paper
, we conducted a review on the appraisal and patient considerations in the use of atorvastatin in the chinese population . to identify all the relative literature involving the use of atorvastatin in the chinese population
, we conducted a systemic search in pubmed with the following keywords : atorvastatin
( supplementary concept ) or atorvastatin ( all field ) and ( china [ ad ] or china [ all field ] or
all clinical or basic research articles on atorvastatin in chinese population were included in this review .
its molecular formula is c33h33cafno5 , with a molecular weight of 582.6947 . like other statins
hmg - coa reductase catalyzes the reduction of hmg - coa to mevalonate , which is the rate - limiting step in cholesterol synthesis in the liver .
meanwhile , the partial depletion of cellular cholesterol by the action of the drugs leads to increasing expression of ldl receptors on hepatocytes .
this increases ldl uptake by the hepatocytes , decreasing the amount of ldl - c in the blood .
inhibition of cholesterol synthesis in hepatocytes results in a decrease in very ldl - c production and an indirect reduction in ldl - c . like other statins
, atorvastatin also reduces blood levels of triglycerides and slightly increases levels of high - density lipoprotein - cholesterol ( hdl - c ) , although the mechanism is still not entirely clear .
the absolute bioavailability of the parent drug is about 14% , but the systemic availability for hmg - coa reductase activity is approximately 30% . the time to reach peak plasma levels ( tmax ) varies from 0.5 to 6 h. administration of atorvastatin with food results in a prolonged tmax , as well as in a reduction in maximum concentration ( cmax ) and area under the curve ( auc ) , however , changes in the rate of atorvastatin absorption are not expected to have a clinically significant effect , as subsequent multiple - dose clinical studies have shown that dose , but not plasma atorvastatin concentration profiles , correlates with lipid - lowering effects . although the rate and the extent of equivalent absorption of atorvastatin were lower during evening than morning administration , the time of administration did not affect the plasma ldl - c - lowering efficacy of atorvastatin .
the major metabolic pathway of atorvastatin is through cytochrome p450 3a4 hydroxylation to form active orthohydroxylated and parahydroxylated metabolites , as well as various beta - oxidation metabolites .
the orthohydroxylated and parahydroxylated metabolites are responsible for 70% of systemic hmg - coa reductase activity . as a substrate for the cyp3a4
, atorvastatin is expected to have drug - drug interaction with concurrent administration of potential inhibitors or inducers of this system . moreover
, several genetic studies have found that cyp gene polymorphisms are associated with the metabolism of atorvastatin in the chinese population and thus affect the lipid - lowering effect of atorvastatin [ table 1 ] .
atorvastatin is primarily eliminated via hepatic biliary excretion without entero - hepatic recirculation and < 2% recovered in the urine .
atorvastatin has an approximate elimination half - life of 14 h. however , the hmg - coa reductase inhibitory activity appears to have a half - life of 2030 h , which is thought to be due to the active metabolites .
gene polymorphisms associated with the metabolism of atorvastatin in chinese population * total cholesterol ; ldl - c : low - density lipoprotein cholesterol . whether there is a differing pattern of systemic exposure to atorvastatin among chinese versus caucasian subjects was studied by gandelman et al . by comparing data obtained from 310 asians ( 31% chinese ) and 572 caucasians in 22 pharmacokinetic studies .
they found that the equivalent dose / bodyweight normalized auc ( asian = 157.5 , caucasian = 156.4 [ nghml]/[mg / kg ] ) and cmax ( asian = 26.2 , caucasian = 30.3 [ ng / ml]/[mg / kg ] ) for atorvastatin were similar in both ethnic groups .
they concluded that dosing considerations in atorvastatin are similar for asian compared with caucasian subjects . a recent meta - analysis of 254 trials evaluating the dose - related efficacy of atorvastatin in 33,505 participants concluded that the blood total cholesterol , ldl - c , and triglyceride - lowering effect of atorvastatin were dependent on dose .
however , there was no significant dose - related effect of atorvastatin on hdl - c . to address
the question of whether the lipid responses to atorvastatin may differ between chinese and caucasians , hu et al .
used the data from the direct statin comparison of ldl - c values : an evaluation of rosuvastatin therapy ( discovery ) program .
they found that there was a similar percentage reduction in ldl - c in response to atorvastatin 10 mg / day in hong kong han chinese patients compared with those in patients from a mixture of other asian or western countries in the discovery program .
the results of the anglo - scandinavian cardiac outcomes trial ( ascot ) showed that hypertensive patients with mildly elevated cholesterol levels who took atorvastatin 10 mg daily had 36% fewer nonfatal myocardial infarction ( mi ) and fatal coronary artery disease than patients treated with placebo . however , ascot only enrolled patients in the united kingdom , ireland , and nordic countries . in a surrogate study from china ,
151 patients with mild hypertensive were randomly divided into three groups : atorvastatin 10 mg group , atorvastatin 20 mg group and the control group .
after 3 months , ldl - c was decreased by 30% in the atorvastatin 10 mg group and 40.48% in the 20 mg group , consistent with the lipid - lowering effect in ascot .
carotid intima media thickness was decreased , and endothelial function was improved in both atorvastatin groups .
a total of 126 hypertensive patients with hypercholesterolemia were randomized into amlodipine 10 mg / day group and amlodipine 10 mg / day plus atorvastatin 20 mg / day group .
after 4 months of treatment with atorvastatin , serum total cholesterol , ldl - c , triglyceride , high sensitive - c reactive protein , and uric acid decreased significantly in the amlodipine plus atorvastatin group , while hdl - c increased ( p < 0.05 ) .
meanwhile , compared with that before treatment , left ventricular mass index in both groups decreased ( p < 0.05 ) , to a significantly lower degree in the amlodipine plus atorvastatin group than in the amlodipine group ( p < 0.05 ) .
type 2 diabetes mellitus ( t2 dm ) is an important risk factor for developing cvd , and it has an atherogenic lipid profile consisting of elevated triglyceride and low hdl - c , which results in high non - hdl - c levels . in the collaborative
atorvastatin diabetes study , 2,838 patients with diabetes aged 4075 years in 132 centers in the united kingdom and ireland were randomized to placebo or atorvastatin 10 mg daily , and atorvastatin 10 mg daily is efficacious in reducing the risk of first cvd events by 37% ( rate reduction 37% , 95% confidence interval [ ci ] - 52 to - 17 ; p = 0.001 ) .
the ascot substudy further confirmed that atorvastatin 10 mg daily significantly reduced the risk of major cv events or procedures ( hazard ratio [ hr ] : 0.77 , 95%ci : 0.610.98 ; p = 0.036 ) among diabetic patients with well - controlled hypertension and without a history of coronary artery disease or markedly elevated cholesterol concentrations .
the proportional reduction in risk was similar to that among participants who did not have diagnosed diabetes .
however , the atorvastatin study for prevention of coronary heart disease endpoints in noninsulin - dependent diabetes mellitus , another rct to access the efficacy of atorvastatin in subjects with t2 dm failed to find a significant reduction in the primary composite end point comparing 10 mg of atorvastatin with placebo in both primary and secondary prevention ( hr : 0.90 , 95%ci : 0.731.12 ; p = 0.34 ) , which may relate to the overall study design , the types of subjects recruited , sample size , and the protocol changes required because of changing treatment guidelines .
recently , su et al . reported data of 151 diabetic patients treated with simvastatin 40 mg / day or atorvastatin 10 mg / day and found that both statins treatment significantly decreased plasma lipids in all patients with t2 dm without a significant difference between the two groups . however , the effects of atorvastatin in increasing nitric oxide concentration and glutathione peroxidase , and superoxide dismutase activity , and in decreasing malondialdehyde level were significantly greater than those of simvastatin in patients with t2 dm compared , suggesting that atorvastatin reduced oxidative stress more effectively than simvastatin did in patients with t2 dm .
liu et al . proved that both atorvastatin 10 mg / day and pravastatin 2 mg / day were effective in reducing the ldl - c level in chinese diabetic patients , as well as in improving insulin resistance and endothelial function .
another study also found that atorvastatin 80 mg / day has a similar effect on endothelial , platelet , and angiogenic indices in both south asians and european with diabetes .
a meta - analysis of 20 rcts investigating different statins has demonstrated that using statins in a high - risk population for primary prevention is associated with a significant reduction in all - cause death , cv death , and major cv events .
this conclusion was confirmed by a recent network meta - analysis , and there is no significant difference between atorvastatin and other statins in primary prevention . however , whether primary prevention is beneficial in individuals at low or modest risk is still uncertain .
the incremental decrease in end points through aggressive lipid lowering trial found that intensive lowering of ldl - c with atorvastatin 80 mg / day did not result in a significant reduction in the primary outcome of major coronary events ( hr : 0.89 , 95%ci : 0.781.01 ; p = 0.07 ) , but did reduce the risk of major cv events ( hr : 0.87 , 95%ci : 0.770.98 ; p = 0.02 ) and nonfatal acute mi ( hr : 0.83 , 95%ci : 0.710.98 ; p = 0.02 ) in patients with previous mi , when compared with usual dose simvastatin ( 20 mg / day ) .
meanwhile in the treating to new targets study , 10,001 patients with stable coronary artery disease were randomized to receive atorvastatin 10 or 80 mg / day to achieve ldl - c goals of < 100 and 70 mg / dl , respectively .
atorvastatin 80 mg / day reduced the risk of cv events to a significantly greater extent than atorvastatin 10 mg / day ( hr : 0.78 , 95%ci : 0.690.89 ; p < 0.001 )
. however , the risk of overall mortality was similar in atorvastatin 80 mg / day recipients , compared with atorvastatin 10 mg / day recipients . in china , 228 patients with stable atherosclerotic plaques who had undergone coronary arteriography and intravascular ultrasound were randomly assigned to receive placebo or atorvastatin at a single daily dose of 10 mg , 20 mg , 40 mg , or 80 mg . at 6 months of follow - up ,
the ldl - c levels in the atorvastatin groups were below those at their respective baselines ( all p < 0.01 ) .
the percentages of plaque necrosis following treatment in the placebo and atorvastatin 10 mg groups rose above baseline levels ( 15.51 12.56 vs. 7.69 1.31% , p < 0.01 ; 13.54 11.76 vs. 7.83 1.43% , p < 0.01 , respectively ) while the atorvastatin 20 , 40 , and 80 mg groups remained stable .
plaque volumes in the atorvastatin 40 and 80 mg groups decreased significantly compared with baseline plaque volumes ( 30.69 8.12 vs. 37.09 12.01 mm , p = 0.019 ; 24.99 1.01 vs. 36.47 14.68 mm , p < 0.01 , respectively ) .
several large rcts have been conducted to assess the efficacy of high - dose atorvastatin in patients with acute coronary syndrome ( acs ) . in the myocardial ischemia reduction with aggressive cholesterol lowering study
, 3,086 patients with unstable angina pectoris or non - q - wave mi were randomized within a mean of 63 h after hospitalization to receive atorvastatin 80 mg / day or placebo for 16 weeks in addition to usual therapy .
atorvastatin therapy reduced the incidence of death , nonfatal acute mi , cardiac arrest with resuscitation , or recurrent symptomatic myocardial ischemia with objective evidence and requiring emergency rehospitalization compared with placebo ( relative risk 0.84 , 95%ci : 0.701.00 ; p = 0.048 ) .
the pravastatin or atorvastatin evaluation and infection therapy ( improve it - timi22 ) study enrolled 4162 patients who had been hospitalized for an acs within the preceding 10 days and compared 40 mg of pravastatin daily ( standard therapy ) with 80 mg of atorvastatin daily ( intensive therapy ) .
after a 2 year follow - up , intensive lipid - lowering statin regimen provides greater protection against death or major adverse cardiac events ( maces ) ( mi , documented unstable angina requiring rehospitalization , revascularization , and stroke ) than a standard regimen ( hr : 0.84 , 95%ci : 0.740.95 , p = 0.005 ) . the efficacy of intensive lipid - lowering with high - dose atorvastatin in acs patients has also been investigated in the chinese population . in the study by dong
92 patients with acs post successful percutaneous coronary intervention ( pci ) were randomly divided into atorvastatin 10 mg / day and atorvastatin 40 mg / day on top of the standard medical therapy for 24 weeks .
total cholesterol and ldl - c in atorvastatin 40 mg / day group were significantly lower than in 10 mg / day group , as were the high sensitive - c reactive protein and matrix metalloproteinases-9 . due to the small sample size
further access the efficacy of high - dose atorvastatin in elderly chinese patient with unstable angina and 166 patients with unstable angina who were 60 years of age , randomly assigned to receive atorvastatin 80 or 20 mg / day .
atorvastatin 80 mg / day was associated with a significant reduction in ldl - c , inflammatory factors , and improvement of endothelial function .
yu et al . found that even a short - term pretreatment with a high dose of atorvastatin ( 80 mg 12 h before pci , with a further 40 mg preprocedure dose ) could reduce the 30 day incidence of maces in chinese patient with non - st elevation acs ( 2.4% vs. 22.5% , p = 0.016 ) , which mainly attributed to the reduction in the incidence of mi ( 2.4% vs. 20.0% , p = 0.031 ) .
however , these studies have several limitations , including small sample size , low study quality , short study duration , and the use of surrogate endpoints instead of clinical endpoints . considering these limitations ,
several large rcts have been initiated to evaluate the efficacy and safety of atorvastatin in chinese patients with coronary artery disease .
the china intensive lipid lowering with statins in acs ( chillas ) study is an open - label multicenter study in china to evaluate whether intensive treatment with statins for 25 years results in a greater reduction of cv events in patients with acs compared with the standard statin therapy .
a total of 1600 patients will be randomly assigned to receive intensive statin therapy ( atorvastatin , 20 or 40 mg / day , or equivalent dose of other statins ) or standard statin therapy ( atorvastatin , 10 mg / day , or equivalent dose of other statins ) .
the primary outcome of the study is the time to occurrence of cardiac death , nonfatal mi , revascularization with either pci or coronary artery bypass surgery , documented unstable angina or severe heart failure requiring emergency hospitalization , and stroke .
the chillas study will be the first multicenter study in a chinese population using a patient - level analysis to compare the effects and safety of intensive statin therapy with that of standard - dose statin therapy .
an intensive statin therapy for chinese patients with coronary artery disease undergoing pci ( iscap ) study is also designed to evaluate the safety and efficacy of intensive statin therapy and the long - term outcome of patients in the chinese population . approximately , 1,100 patients with stable angina or non - st elevation acs undergoing selective pci will be enrolled and randomized either to the intensive group ( atorvastatin 80 mg / day 2 days before and 40 mg / day 30 days after pci ) or to the control group ( usual care ) .
the primary outcome is 30 day maces , and secondary outcomes are 6 months maces and change in biomarkers .
the result of the iscap study will provide important evidence on the efficacy and safety of periprocedural serial intensive statin treatment in chinese patients with coronary artery disease undergoing selective pci .
newman et al . conducted a pooled analysis from 44 clinical trials comprising 16,495 dyslipidemic patients to access the safety , tolerability , and adherence of atorvastatin in the 10 mg / day to 80 mg / day dose range . in this study , only 3% ( n = 241 ) of atorvastatin - treated patients withdrew from studies due to treatment - associated adverse events , compared with 1% of those ( n = 16 ) on placebo and 4% of those ( n = 188 ) receiving other statins .
the most frequently reported treatment - associated adverse events were related to the digestive system .
elevations in alanine aminotransferase ( alt ) were similar for both the atorvastatin and the other statin treatment groups relative to the placebo group
. persistent alt to > 3 times the upper limit of normal ( uln ) were experienced by 0.5% ( n = 47 ) of atorvastatin - treated patients , compared with 1% of those ( n = 16 ) on placebo and 4% of those ( n = 188 ) receiving other statins . a persistent elevation in creatine phosphokinase ( ck ) ( > 10 uln )
was observed in only one atorvastatin - treated patient and was not associated with myopathy .
the incidence of treatment - associated myalgia was low in the atorvastatin ( 1.9% [ n = 181 ] ) , placebo ( 0.8% [ n = 14 ] ) , and other statin ( 2.0% [ n = 105 ] ) groups , and was not related to the atorvastatin dose .
the overall incidence of treatment - associated adverse events observed with atorvastatin did not increase in the 1080 mg dose range and was similar to that observed with placebo and in patients treated with other statins .
specific analysis of musculoskeletal and hepatic adverse events showed that these infrequently occurred and rarely resulted in treatment discontinuation .
an updated pooled analysis of 49 clinical trials comparing the safety of atorvastatin 10 mg / day , atorvastatin 80 mg / day , and placebo further confirm that the incidence of treatment - associated adverse events for atorvastatin 80 mg / day was similar to that of atorvastatin 10 mg / day and placebo , as well as the incidence withdrawals due to treatment - related adverse events . to fully evaluate the safety and adherence of atorvastatin in the chinese population , we conducted a systemic search of the literature in pubmed using the keywords atorvastatin
( supplementary concept ) or atorvastatin ( all field ) and ( china [ ad ] or china [ all field ] or
[ all field ] ) to identify all the rcts comparing atorvastatin with control / placebo or other statins and reporting the incidence of side - effect .
the incidence of hepatic and musculoskeletal toxicity and the adherence of medication were pooled using the mantel - haenszel method with random effect model ( stata 11.0 ; stata corp .
the risk of hepatic and musculoskeletal toxicity did not differ between atorvastatin and control / placebo groups , as well as the adherence of medication .
besides , atorvastatin has a similar risk of hepatic and musculoskeletal toxicity and adherence of medication with other statins . when compared with low - dose atorvastatin ( 1020 mg / day ) , high - dose atorvastatin ( 4080 mg / day )
however , the risk of elevated alt > 3 uln , as well as the risk of musculoskeletal toxicity and adherence to medications , did not differ between high - dose and low - dose atorvastatin . thus , the overall incidence of significant adverse events observed with atorvastatin in chinese population was similar to that observed with placebo or other statins [ figure 1 ] .
safety outcomes and patients adherence of atorvastatin in randomized controlled trials with chinese population * acute coronary syndrome ; coronary artery disease ; percutaneous coronary intervention ; unstable angina ; high sensitive c reactive protein ; matrix metalloproteinases ; * * carotid intima thickness ; major adverse cardiac event ; loading dose ; maintain dose ; alanine aminotransferase ; upper limit of normal ; * * * creatine phosphokinase ; not available .
recently , statin therapy was found to be associated with an increased risk of development of diabetes , and it is believed that different types and doses of statins show different potential to increase the incidence of diabetes . compared with pravastatin , treatment with higher potency statins , especially atorvastatin and simvastatin , might be associated with an increased risk of new - onset diabetes .
however , a retrospective cohort study of 15,637 chinese hypertensive and dyslipidemic patients demonstrated that patients who take atorvastatin or rosuvastatin are at lower risk of new - onset diabetes , while lovastatin and simvastatin were associated with a significant increase in the risk of new - onset diabetes . compared with lower - dose atorvastatin , atorvastatin 80 mg / day
did not increase the incidence of new - onset diabetes in patients with 0 to 1 risk factors but did , by 24% , among patients with two to four risk factors for new - onset diabetes . of note
, the risk of new - onset diabetes is low both in absolute terms and when compared with a reduction in coronary events .
first , only few studies were designed to compare the difference in use of atorvastatin directly between chinese and western population .
second , high - quality evidence on atorvastatin , as well as the other statins , remained unavailable in chinese population .
a recent meta - analysis of 254 trials evaluating the dose - related efficacy of atorvastatin in 33,505 participants concluded that the blood total cholesterol , ldl - c , and triglyceride - lowering effect of atorvastatin were dependent on dose .
however , there was no significant dose - related effect of atorvastatin on hdl - c . to address the question of whether the lipid responses to atorvastatin may differ between chinese and caucasians , hu et al
. used the data from the direct statin comparison of ldl - c values : an evaluation of rosuvastatin therapy ( discovery ) program .
they found that there was a similar percentage reduction in ldl - c in response to atorvastatin 10 mg / day in hong kong han chinese patients compared with those in patients from a mixture of other asian or western countries in the discovery program .
the results of the anglo - scandinavian cardiac outcomes trial ( ascot ) showed that hypertensive patients with mildly elevated cholesterol levels who took atorvastatin 10 mg daily had 36% fewer nonfatal myocardial infarction ( mi ) and fatal coronary artery disease than patients treated with placebo . however , ascot only enrolled patients in the united kingdom , ireland , and nordic countries . in a surrogate study from china ,
151 patients with mild hypertensive were randomly divided into three groups : atorvastatin 10 mg group , atorvastatin 20 mg group and the control group . after 3 months , ldl - c
was decreased by 30% in the atorvastatin 10 mg group and 40.48% in the 20 mg group , consistent with the lipid - lowering effect in ascot .
carotid intima media thickness was decreased , and endothelial function was improved in both atorvastatin groups .
a total of 126 hypertensive patients with hypercholesterolemia were randomized into amlodipine 10 mg / day group and amlodipine 10 mg / day plus atorvastatin 20 mg / day group .
after 4 months of treatment with atorvastatin , serum total cholesterol , ldl - c , triglyceride , high sensitive - c reactive protein , and uric acid decreased significantly in the amlodipine plus atorvastatin group , while hdl - c increased ( p < 0.05 ) .
meanwhile , compared with that before treatment , left ventricular mass index in both groups decreased ( p < 0.05 ) , to a significantly lower degree in the amlodipine plus atorvastatin group than in the amlodipine group ( p < 0.05 ) .
type 2 diabetes mellitus ( t2 dm ) is an important risk factor for developing cvd , and it has an atherogenic lipid profile consisting of elevated triglyceride and low hdl - c , which results in high non - hdl - c levels . in the collaborative atorvastatin diabetes study , 2,838 patients with diabetes aged 4075 years in 132 centers in the united kingdom and ireland were randomized to placebo or atorvastatin 10 mg daily , and atorvastatin 10 mg daily is efficacious in reducing the risk of first cvd events by 37% ( rate reduction 37% , 95% confidence interval [ ci ] - 52 to - 17 ; p = 0.001 ) .
the ascot substudy further confirmed that atorvastatin 10 mg daily significantly reduced the risk of major cv events or procedures ( hazard ratio [ hr ] : 0.77 , 95%ci : 0.610.98 ; p = 0.036 ) among diabetic patients with well - controlled hypertension and without a history of coronary artery disease or markedly elevated cholesterol concentrations .
the proportional reduction in risk was similar to that among participants who did not have diagnosed diabetes .
however , the atorvastatin study for prevention of coronary heart disease endpoints in noninsulin - dependent diabetes mellitus , another rct to access the efficacy of atorvastatin in subjects with t2 dm failed to find a significant reduction in the primary composite end point comparing 10 mg of atorvastatin with placebo in both primary and secondary prevention ( hr : 0.90 , 95%ci : 0.731.12 ; p = 0.34 ) , which may relate to the overall study design , the types of subjects recruited , sample size , and the protocol changes required because of changing treatment guidelines . none of these three studies included chinese population . recently , su et al .
reported data of 151 diabetic patients treated with simvastatin 40 mg / day or atorvastatin 10 mg / day and found that both statins treatment significantly decreased plasma lipids in all patients with t2 dm without a significant difference between the two groups .
however , the effects of atorvastatin in increasing nitric oxide concentration and glutathione peroxidase , and superoxide dismutase activity , and in decreasing malondialdehyde level were significantly greater than those of simvastatin in patients with t2 dm compared , suggesting that atorvastatin reduced oxidative stress more effectively than simvastatin did in patients with t2 dm .
liu et al . proved that both atorvastatin 10 mg / day and pravastatin 2 mg / day were effective in reducing the ldl - c level in chinese diabetic patients , as well as in improving insulin resistance and endothelial function .
another study also found that atorvastatin 80 mg / day has a similar effect on endothelial , platelet , and angiogenic indices in both south asians and european with diabetes .
a meta - analysis of 20 rcts investigating different statins has demonstrated that using statins in a high - risk population for primary prevention is associated with a significant reduction in all - cause death , cv death , and major cv events .
this conclusion was confirmed by a recent network meta - analysis , and there is no significant difference between atorvastatin and other statins in primary prevention . however , whether primary prevention is beneficial in individuals at low or modest risk is still uncertain .
the incremental decrease in end points through aggressive lipid lowering trial found that intensive lowering of ldl - c with atorvastatin 80 mg / day did not result in a significant reduction in the primary outcome of major coronary events ( hr : 0.89 , 95%ci : 0.781.01 ; p = 0.07 ) , but did reduce the risk of major cv events ( hr : 0.87 , 95%ci : 0.770.98 ; p = 0.02 ) and nonfatal acute mi ( hr : 0.83 , 95%ci : 0.710.98 ; p = 0.02 ) in patients with previous mi , when compared with usual dose simvastatin ( 20 mg / day ) .
meanwhile in the treating to new targets study , 10,001 patients with stable coronary artery disease were randomized to receive atorvastatin 10 or 80 mg / day to achieve ldl - c goals of < 100 and 70 mg / dl , respectively .
atorvastatin 80 mg / day reduced the risk of cv events to a significantly greater extent than atorvastatin 10 mg / day ( hr : 0.78 , 95%ci : 0.690.89 ; p < 0.001 ) .
however , the risk of overall mortality was similar in atorvastatin 80 mg / day recipients , compared with atorvastatin 10 mg / day recipients . in china , 228 patients with stable atherosclerotic plaques who had undergone coronary arteriography and intravascular ultrasound were randomly assigned to receive placebo or atorvastatin at a single daily dose of 10 mg , 20 mg , 40 mg , or 80 mg . at 6 months of follow - up
, the ldl - c levels in the atorvastatin groups were below those at their respective baselines ( all p < 0.01 ) .
the percentages of plaque necrosis following treatment in the placebo and atorvastatin 10 mg groups rose above baseline levels ( 15.51 12.56 vs. 7.69 1.31% , p < 0.01 ; 13.54 11.76 vs. 7.83 1.43% , p < 0.01 , respectively ) while the atorvastatin 20 , 40 , and 80 mg groups remained stable .
plaque volumes in the atorvastatin 40 and 80 mg groups decreased significantly compared with baseline plaque volumes ( 30.69 8.12 vs. 37.09 12.01 mm , p = 0.019 ; 24.99 1.01 vs. 36.47 14.68 mm , p < 0.01 , respectively ) .
several large rcts have been conducted to assess the efficacy of high - dose atorvastatin in patients with acute coronary syndrome ( acs ) . in the myocardial ischemia reduction with aggressive cholesterol lowering study , 3,086 patients with unstable angina pectoris or non - q - wave mi were randomized within a mean of 63 h after hospitalization to receive atorvastatin 80 mg / day or placebo for 16 weeks in addition to usual therapy .
atorvastatin therapy reduced the incidence of death , nonfatal acute mi , cardiac arrest with resuscitation , or recurrent symptomatic myocardial ischemia with objective evidence and requiring emergency rehospitalization compared with placebo ( relative risk 0.84 , 95%ci : 0.701.00 ; p = 0.048 ) .
the pravastatin or atorvastatin evaluation and infection therapy ( improve it - timi22 ) study enrolled 4162 patients who had been hospitalized for an acs within the preceding 10 days and compared 40 mg of pravastatin daily ( standard therapy ) with 80 mg of atorvastatin daily ( intensive therapy ) .
after a 2 year follow - up , intensive lipid - lowering statin regimen provides greater protection against death or major adverse cardiac events ( maces ) ( mi , documented unstable angina requiring rehospitalization , revascularization , and stroke ) than a standard regimen ( hr : 0.84 , 95%ci : 0.740.95 , p = 0.005 ) .
the efficacy of intensive lipid - lowering with high - dose atorvastatin in acs patients has also been investigated in the chinese population . in the study by dong et al .
, 92 patients with acs post successful percutaneous coronary intervention ( pci ) were randomly divided into atorvastatin 10 mg / day and atorvastatin 40 mg / day on top of the standard medical therapy for 24 weeks .
total cholesterol and ldl - c in atorvastatin 40 mg / day group were significantly lower than in 10 mg / day group , as were the high sensitive - c reactive protein and matrix metalloproteinases-9 . due to the small sample size
further access the efficacy of high - dose atorvastatin in elderly chinese patient with unstable angina and 166 patients with unstable angina who were 60 years of age , randomly assigned to receive atorvastatin 80 or 20 mg / day .
atorvastatin 80 mg / day was associated with a significant reduction in ldl - c , inflammatory factors , and improvement of endothelial function .
yu et al . found that even a short - term pretreatment with a high dose of atorvastatin ( 80 mg 12 h before pci , with a further 40 mg preprocedure dose ) could reduce the 30 day incidence of maces in chinese patient with non - st elevation acs ( 2.4% vs. 22.5% , p = 0.016 ) , which mainly attributed to the reduction in the incidence of mi ( 2.4% vs. 20.0% , p = 0.031 ) .
however , these studies have several limitations , including small sample size , low study quality , short study duration , and the use of surrogate endpoints instead of clinical endpoints . considering these limitations ,
several large rcts have been initiated to evaluate the efficacy and safety of atorvastatin in chinese patients with coronary artery disease .
the china intensive lipid lowering with statins in acs ( chillas ) study is an open - label multicenter study in china to evaluate whether intensive treatment with statins for 25 years results in a greater reduction of cv events in patients with acs compared with the standard statin therapy .
a total of 1600 patients will be randomly assigned to receive intensive statin therapy ( atorvastatin , 20 or 40 mg / day , or equivalent dose of other statins ) or standard statin therapy ( atorvastatin , 10 mg / day , or equivalent dose of other statins ) .
the primary outcome of the study is the time to occurrence of cardiac death , nonfatal mi , revascularization with either pci or coronary artery bypass surgery , documented unstable angina or severe heart failure requiring emergency hospitalization , and stroke .
the chillas study will be the first multicenter study in a chinese population using a patient - level analysis to compare the effects and safety of intensive statin therapy with that of standard - dose statin therapy .
an intensive statin therapy for chinese patients with coronary artery disease undergoing pci ( iscap ) study is also designed to evaluate the safety and efficacy of intensive statin therapy and the long - term outcome of patients in the chinese population .
approximately , 1,100 patients with stable angina or non - st elevation acs undergoing selective pci will be enrolled and randomized either to the intensive group ( atorvastatin 80 mg / day 2 days before and 40 mg / day 30 days after pci ) or to the control group ( usual care ) .
the primary outcome is 30 day maces , and secondary outcomes are 6 months maces and change in biomarkers .
the result of the iscap study will provide important evidence on the efficacy and safety of periprocedural serial intensive statin treatment in chinese patients with coronary artery disease undergoing selective pci .
newman et al . conducted a pooled analysis from 44 clinical trials comprising 16,495 dyslipidemic patients to access the safety , tolerability , and adherence of atorvastatin in the 10 mg / day to 80 mg / day dose range . in this study , only 3% ( n = 241 ) of atorvastatin - treated patients withdrew from studies due to treatment - associated adverse events , compared with 1% of those ( n = 16 ) on placebo and 4% of those ( n = 188 ) receiving other statins .
the most frequently reported treatment - associated adverse events were related to the digestive system .
elevations in alanine aminotransferase ( alt ) were similar for both the atorvastatin and the other statin treatment groups relative to the placebo group
. persistent alt to > 3 times the upper limit of normal ( uln ) were experienced by 0.5% ( n = 47 ) of atorvastatin - treated patients , compared with 1% of those ( n = 16 ) on placebo and 4% of those (
n = 188 ) receiving other statins . a persistent elevation in creatine phosphokinase ( ck ) ( > 10 uln )
was observed in only one atorvastatin - treated patient and was not associated with myopathy .
the incidence of treatment - associated myalgia was low in the atorvastatin ( 1.9% [ n = 181 ] ) , placebo ( 0.8% [ n = 14 ] ) , and other statin ( 2.0% [ n = 105 ] ) groups , and was not related to the atorvastatin dose .
the overall incidence of treatment - associated adverse events observed with atorvastatin did not increase in the 1080 mg dose range and was similar to that observed with placebo and in patients treated with other statins .
specific analysis of musculoskeletal and hepatic adverse events showed that these infrequently occurred and rarely resulted in treatment discontinuation .
an updated pooled analysis of 49 clinical trials comparing the safety of atorvastatin 10 mg / day , atorvastatin 80 mg / day , and placebo further confirm that the incidence of treatment - associated adverse events for atorvastatin 80 mg / day was similar to that of atorvastatin 10 mg / day and placebo , as well as the incidence withdrawals due to treatment - related adverse events . to fully evaluate the safety and adherence of atorvastatin in the chinese population
, we conducted a systemic search of the literature in pubmed using the keywords atorvastatin
( supplementary concept ) or atorvastatin ( all field ) and ( china [ ad ] or china [ all field ] or
[ all field ] ) to identify all the rcts comparing atorvastatin with control / placebo or other statins and reporting the incidence of side - effect .
the incidence of hepatic and musculoskeletal toxicity and the adherence of medication were pooled using the mantel - haenszel method with random effect model ( stata 11.0 ; stata corp . ,
the risk of hepatic and musculoskeletal toxicity did not differ between atorvastatin and control / placebo groups , as well as the adherence of medication .
besides , atorvastatin has a similar risk of hepatic and musculoskeletal toxicity and adherence of medication with other statins . when compared with low - dose atorvastatin ( 1020 mg / day ) , high - dose atorvastatin ( 4080 mg / day ) has a significantly higher risk of elevated alt .
however , the risk of elevated alt > 3 uln , as well as the risk of musculoskeletal toxicity and adherence to medications , did not differ between high - dose and low - dose atorvastatin .
thus , the overall incidence of significant adverse events observed with atorvastatin in chinese population was similar to that observed with placebo or other statins [ figure 1 ] .
safety outcomes and patients adherence of atorvastatin in randomized controlled trials with chinese population * acute coronary syndrome ; coronary artery disease ; percutaneous coronary intervention ; unstable angina ; high sensitive c reactive protein ; matrix metalloproteinases ; * * carotid intima thickness ; major adverse cardiac event ; loading dose ; maintain dose ; alanine aminotransferase ; upper limit of normal ; * * * creatine phosphokinase ; not available .
recently , statin therapy was found to be associated with an increased risk of development of diabetes , and it is believed that different types and doses of statins show different potential to increase the incidence of diabetes .
compared with pravastatin , treatment with higher potency statins , especially atorvastatin and simvastatin , might be associated with an increased risk of new - onset diabetes .
however , a retrospective cohort study of 15,637 chinese hypertensive and dyslipidemic patients demonstrated that patients who take atorvastatin or rosuvastatin are at lower risk of new - onset diabetes , while lovastatin and simvastatin were associated with a significant increase in the risk of new - onset diabetes . compared with lower - dose atorvastatin , atorvastatin
80 mg / day did not increase the incidence of new - onset diabetes in patients with 0 to 1 risk factors but did , by 24% , among patients with two to four risk factors for new - onset diabetes . of note ,
the risk of new - onset diabetes is low both in absolute terms and when compared with a reduction in coronary events .
first , only few studies were designed to compare the difference in use of atorvastatin directly between chinese and western population .
second , high - quality evidence on atorvastatin , as well as the other statins , remained unavailable in chinese population .
although the benefit of atorvastatin in primary and secondary prevention has been verified by abundant of international multiple - center rcts , none of them included the chinese population , and the evidence of atorvastatin in the chinese population is still insufficient .
however , based on the current available evidence , there is no significant difference between the chinese and non - chinese populations in terms of pharmacology and clinical efficacy .
thus , atorvastatin still should be considered as one of the first - line medication in chinese patients with lipid disorders .
however , high - quality evidence is needed to support the use of atorvastatin in high - risk chinese population . | objective : statins are still underused for the prevention of cardiovascular disease ( cvd ) in china .
hence , we conducted a systemic review on the pharmacology , clinical efficacy , and adverse events of atorvastatin , as well as on patient adherence.data sources : we conducted a systemic search in pubmed with the following keywords : atorvastatin
( supplementary concept ) or atorvastatin ( all field ) and ( china [ ad ] or china [ all field ] or
chinese
[ all field]).study selection : clinical or basic research articles on atorvastatin were included.results:atorvastatin is a reversible and competitive inhibitor of 3-hydroxy-3-methylglutaryl - coenzyme a reductase , decreasing the de novo cholesterol synthesis .
the pharmacokinetics of atorvastatin among chinese is similar to those in caucasians , and several gene polymorphisms have proved to be associated with the metabolism of atorvastatin in the chinese population .
several international multiple - center randomized control trials have demonstrated the benefit of atorvastatin for primary and secondary prevention of cvd .
none of them , however , included the chinese , and current evidence in the population is still inadequate , due to the small sample size , low study quality , short study duration , and the use of surrogate endpoints instead of clinical endpoints .
the overall incidence of adverse events observed with atorvastatin did not increase in the 1080 mg dose range , and was similar to that observed with placebo and in patients treated with other statins , which makes atorvastatin well - tolerated in the chinese population .
moreover , high patient adherence was observed in clinical studies.conclusions:based on the current available evidence , there is no significant difference between chinese and non - chinese population in term of pharmacology and clinical efficacy / safety .
high - quality evidence is still needed to support the use of atorvastatin in high - risk chinese population . |
cancer of the nasal cavity and the paranasal sinuses , referred to as sinonasal cancer ( snc ) , is relatively uncommon in the general population , accounting for less than 1% of all neoplasms and less than 4% of those arising in the head and neck region [ 13 ] .
snc incidence is around 1 100,000 person - years in most developed countries . in the period 19982002 ,
the annual incidence rates in the united states were 0.8 and 0.6 100,000 in males and females , respectively .
in the same period , the rarecare project , based on about 2200 cases of epithelial tumors of nasal cavities in europe , showed an age - standardized rate of 0.36 100,000 .
the incidence rates in 19982002 estimated by the italian network of cancer registries ( associazione italiana registri tumori , airtum , http://www.registri-tumori.it/ ) , which covers 40% of the whole population , were between 0.4 and 2.0 in males and 0.1 and 0.5 100,000 in females , with about 300 cases expected per year in the whole country ; a high variability across italian regions was reported .
frequency , anatomical site , and histological type of sncs vary across geographical areas due to several factors .
a history of chronic sinusitis , nasal polyps , use of nasal drug preparations , and smoking and occupational history of wood and leather working and nickel refining are reported as risk factors for the development of these tumors . a strong relationship between snc and exposure to wood , leather dust , and nickel compounds
other confirmed or suspected causative factors include hexavalent chromium compounds , welding fumes , arsenic , mineral oils , organic solvents , and textile dust [ 11 , 1316 ] .
for this reason , in italy , since 2008 , their incidence and etiology are compulsorily surveyed in the whole italian population ( about 60 million people ) through a nationwide cancer registry ( renatuns ) , coordinated by the italian workers ' compensation authority ( istituto nazionale per l'assicurazione contro gli infortuni sul lavoro , inail ) .
renatuns collects information from a network of regional registries to monitor snc incidence and to establish sources of occupational exposure ( http://www.inail.it/ ) . in the lombardy region , northwest italy , the most populated ( almost 10 million residents at the 2011 census ) and industrialized italian region ,
we present clinical characteristics and sources of occupational exposure for snc cases recorded in the period 20082011 .
we also calculated lombardy incidence rates of snc for the period 2008 - 2009 , in which registration of clinical and exposure information was complete .
the lombardy registry of sinonasal cancers ( lrsnc ) collects all snc cases occurring among subjects with residence in the region ( from about 9.6 million people in 2008 to about 9.9 million in 2011 ) at the time of first diagnosis .
the primary sources of information on snc cases are the departments of diagnosis and treatment of snc in regional hospitals ( more than 100 ) , particularly pathology , otolaryngology , maxillofacial surgery , and radiotherapy .
completeness of reporting is checked by periodic linkage made at lrsnc with databases of pathology departments ( six - monthly ) , hospital discharge records ( annual ) , mortality registries ( annual ) , and occupational disease compensation records from inail ( annual ) .
this complex system virtually ensures complete ascertainment of snc cases occurring in lombardy residents and also allows the identification of the few patients admitted in hospitals outside the lombardy region .
diagnosis of snc is established by a panel of experts at lrsnc based on complete clinical information ( copy of clinical records , computed tomography and/or magnetic resonance imaging scans , pathology reports ) according to the renatuns guidelines .
snc cases include all newly diagnosed primary malignant epithelial cancers of the nasal cavity , code c30.0 of the international classification of diseases , tenth revision ( icd-10 ) and paranasal sinus , icd-10 codes from c31.0 to c31.9 .
a standardized questionnaire is administered to patients or their next of kin by trained interviewers within the hospital in which diagnosis was made or at the occupational health services of the local health units in lombardy region .
the questionnaire is designed to obtain a lifetime occupational history , including industrial sectors , plants , jobs , and specific task performed .
coding of the occupational history is performed according to the italian classification of economic activities .
the questionnaire is then reviewed by occupational physicians and industrial hygienists who assess occupational exposure to agents with an established or suspected association with snc : wood and leather dust , nickel and chromium compounds , polycyclic aromatic hydrocarbons ( pah ) , and cork dust .
also exposure to these agents in domestic setting or during hobby activities is evaluated . in case of exposure either in occupational or in other settings ,
the subjects is classified as occupationally exposed . for the period 20082011 , to compare the distribution of demographics , clinical characteristics , and occupational exposures among snc cases across gender and tumor morphology , we used the chi - square test .
age distribution across gender was evaluated using mann - whitney u test . for the period 2008 - 2009 , in which case registration was complete , we calculated age - standardized rates of snc ( 100,000 person - years ) separately for men and women using as standards either the italian population in the year 2001 or the european population
. confidence intervals ( ci ) of standardized rates were calculated according to the formula proposed by tiwari et al . [ 19 , 20 ] .
in the period 20082011 , we recorded 210 snc cases , 137 ( 65.2% ) among males and 73 ( 34.8% ) among females ( table 1 ) .
about one - third of the tumors originated from the nasal cavity , one - fourth from the maxillary sinus , and one - fifth from multiple sites .
the ethmoid sinus was affected in almost 25% of men and only about 7% of women .
the questionnaire was administered directly to the patient in 146 cases ( 69.5% ) and to his / her relatives in 56 ( 26.6% ) .
smokers were more frequent in subjects with squamous cell carcinoma ( 63.9% ) or adenocarcinoma ( 68.3% ) than in subjects with other morphologies ( 56.1% ) .
current smokers were more frequent among subjects with squamous cell carcinomas ( 34.9% ) than in subjects with adenocarcinoma ( 16.7% ) or with other morphologies ( 15.8% ) .
a history of nasal polyps was reported by 18 subjects ( 8.6% ) , 15 men and 3 women . in the years 2008 - 2009
, we recorded 82 snc cases among men and 45 among women . in these years
, registration was completed , while for the following two years ( 55 cases in men and 28 in women ) , completeness checks are still in progress .
using the italian 2001 population as standard , in the years 2008 - 2009 , the age - standardized rates ( 100,000 person - years ) of snc were 0.9 ( 95% ci : 0.71.1 ) in men and 0.4 ( 95% ci : 0.30.5 ) in women . using the european population as standard
, the snc incidence rates were 0.7 ( 95% ci : 0.50.8 ) in men and 0.3 ( 95% ci : 0.20.4 ) in women .
age - specific rates had a peak over 60 years of age in both genders ; however , rates began to increase at lower ages , especially in men ( figure 1 ) . among men , occupational exposure to any of the six carcinogenic agents mentioned above was identified in 66 subjects ( 48.2% ) , while among women , the proportion was 15.1% ( table 2 ) .
wood and leather dust particles were the most prevalent exposures , especially among males ( 28.5% and 16.8% of subjects , resp . ) . in women , the corresponding proportions were 6.9% and 8.2% .
seven subjects ( six men ) had been ever exposed to wood dust and another agent : leather dust ( four ) , nickel compounds ( two ) , and chromium compounds ( one subject ) .
we also recorded extraoccupational exposure to a carcinogen for 8 subjects . in the first case
, the subject had been exposed to leather dust in the domestic setting , where the mother produced leather buttons .
the other seven cases had been exposed to wood dust during hobby activities ( construction of furniture and wooden objects or hobby modeling ) , without using personal protective equipment . among subjects with adenocarcinoma ,
occupational exposure was recorded for the majority ( 77.4% ) of subjects ( table 3 ) .
half had been ever exposed to wood dust and almost one - third to leather dust .
one case was exposed to cork dust . among subjects with squamous cell carcinoma and with tumors of other morphologies ,
the proportions of subjects ever exposed to any of the six carcinogens were 17.0% and 23.3% , respectively .
exposure to wood or leather dust was about 10% in the case of squamous cell carcinoma and about 20% for other morphologies .
wood dust exposure occurred mainly ( about 80% ) in the wood and furniture industry , followed by building construction sectors ( 8% ) .
the rest of the subjects were exposed in the metal - mechanic sector ( construction of wood patterns , use of sawdust in metal polishing ) or in a turkey farm ( use of sawdust as bedding ) .
leather dust exposure occurred mainly in boot and shoe manufacture and repair ( 90% ) .
exposure to nickel compounds was recorded in the electroplating industry and in the paint manufacture .
exposure to hexavalent chromium was found for car painters using chromium paints and in the printing industry ( use of chromic acid ) .
the mean length of occupational exposure was 26.6 years ( range : 1 to 63 ) in the 66 men and 15.3 ( 1 to 38 ) in the 11 women .
the mean latency was 53.3 years ( min 18.2 , max 74.4 ) in men and 42.1 years ( min 13.6 , max 74.6 ) in women . occupational exposure to any of the six carcinogens was relatively more frequent ( 51.3% ) among former smokers ( table 4 ) .
occupational exposure was similarly distributed among the 18 subjects with a history of nasal polyps ( 8 cases , 44.4% ) and among the 192 without ( 69 cases , 36.9% ) ( p value = 0.47 ) .
this study describes the findings of lrsnc , a dedicated snc regional registry . for the first time ,
incidence of sncs and the role of exposure to recognized carcinogens were examined in an italian large population ( nearly 10 million people ) , in a region where shoe / boot factories and repair shops , furniture production , and smelting and metal - mechanic industries are still widely present .
incidence rates in men were more than twice as high as in women , probably reflecting differences in previous exposure to carcinogens . in this population - wide case series
, we found that tumors originate more often ( one - third ) in the nasal cavities , while localization in maxillary ( one - fourth ) and ethmoid sinuses ( almost one - fifth ) was less frequent .
this is in agreement with a recent review which considered us and italian cancer registries data ( 19982002 ) , with a report from the netherlands registry covering the period 19732009 , and with uk estimates for 2012 .
however , in about 20% of cases , we were not able to determine the site of tumor 's origin because multiple sites were involved at first diagnosis .
again , this is in agreement with the mentioned us , italian , dutch , and british studies [ 2 , 22 , 23 ] . in our study
, we found a closer association between smoking and squamous cell carcinomas and male gender .
the literature estimates of the percentage of sncs attributable to occupational exposures range from 25% to 41% . in our study , the attributable fraction was 38.5% . in men
the proportion of subjects ever exposed occupationally was almost 50% , but also in women it was not negligible ( 15% ) .
this finding underlines the need to investigate the exposure history , including occupation , of each case of snc .
we found a high frequency of exposure to wood and leather dust , especially among men and among subjects with adenocarcinoma . although lower , the proportion of subjects with other histology ( squamous cell carcinoma or other morphologies ) exposed to wood or leather dust was not negligible ( about 1020% ) .
this suggests that not only adenocarcinoma of ethmoid sinus can be induced by wood and leather dust exposure [ 17 , 26 ] .
first , registration of cases in the lrsnc is quite complete , because the reporting of snc cases by departments of diagnosis and treatment of snc is supplemented by an active research which exploits all available information systems ( databases of pathology departments , hospital discharge records , mortality registries , and occupational disease compensation records ) .
for this reason , we are confident that the number of cases not identified through these sources is virtually zero .
third , diagnostic evaluation is made blind of exposure ( the interview is performed after the diagnosis has been verified ) .
the major limitation lies in the fact that the years 2010 - 2011 are still incomplete because some sources of data become available late after case occurrence .
therefore , the exposure profile of the current ( in progress ) case series might in theory be different from that calculated in the complete case series .
however , when we restricted the analysis to the period 2008 - 2009 , the exposure profile was quite similar to that observed in the whole 20082011 period ( results not shown ) .
in conclusion , our study shows that complete collection of cases through a dedicated cancer registry can provide a clear picture of snc occurrence in a population and of exposure to several relevant carcinogens , in different industrial sectors , including those not considered as posing a high risk of snc , and also in extra - occupational settings . | we examined occupational exposures among subjects with sinonasal cancer ( snc ) recorded in a population - based registry in the lombardy region , the most populated and industrialized italian region .
the registry collects complete clinical information and exposure to carcinogens regarding all snc cases occurring in the population of the region . in the period 20082011
, we recorded 210 snc cases ( 137 men , 73 women ) .
the most frequent occupational exposures were to wood ( 44 cases , 21.0% ) and leather dust ( 29 cases , 13.8% ) , especially among men : 39 cases ( 28.5% ) to wood and 23 cases ( 16.8% ) to leather dust .
exposure to other agents was infrequent ( < 2% ) . among 62 subjects with adenocarcinoma
, 50% had been exposed to wood dust and 30.7% to leather dust .
the proportions were around 10% in subjects with squamous cell carcinoma and about 20% for tumors with another histology .
the age - standardized rates ( 100,000 person - years ) were 0.7 in men and 0.3 in women .
complete collection of cases and their occupational history through a specialized cancer registry is fundamental to accurately monitor snc occurrence in a population and to uncover exposure to carcinogens in different industrial sectors , even those not considered as posing a high risk of snc , and also in extraoccupational settings . |
maintaining or increasing soil organic carbon ( soc ) storage in croplands has been promoted as a win
win strategy by enhancing soil productivity and increasing the terrestrial c stock , thereby mitigating greenhouse gas ( ghg ) emissions in agriculture ( lal 2004a ) .
crop management may have great potential for mitigating climate change in agriculture through c sequestration ( smith et al .
this potential , however , will be realized only in the long - run through judicious land use and recommended management practices ( lal 2002 , 2004a ) , whereas the choice among these may vary with the market c price ( lewandrowski et al .
management practices that lead to an increase in organic inputs ( biomass and manure ) add c to the soil , improve microbial function and promote soc accumulation .
it is well known that improved crop management , such as reduced tillage and intensified cropping to enhance c input to the soil , favours soc sequestration ( lal 2004a ; ogle et al .
land use changes and management , however , have significant effects on world soc storage and sequestration for potential mitigation of global climate change ( guo & gifford 2002 ; schlamadinger et al .
2007 ;
there have been a few studies dealing with changes in soc storage under land use and management change in croplands ( osher et al .
2003 ; smith 2008 ) . in developing countries , constraints on land resource availability due to fragmented croplands and small - scale farm management systems impact on sustainable agriculture and food security ( simmons 1986
soil organic matter has generally been recognized as providing a key soil function for sustainable agriculture ( smith et al .
1999 ; carter 2002 ) , and may be influenced by changes in land use intensity and land management effects ( grandy & robertson 2007 ) . the potential roles of improved land use and management in enhancing c stocks in agricultural soils for developing countries have often been addressed ( garca - oliva & masera 2004 ; milne et al .
2007 ;
however , a number of land policy and management barriers will probably prevent soil c sequestration benefits for developing countries , and proper policies for world climate and trade are required for encouraging c sequestration and climate change mitigation in agriculture in less developed countries ( de costa & sangakkara 2006 ; smith & trines 2007 ; eitzinger et al .
c sequestration in croplands of developing countries will depend on the existing performance of these management practices as implemented by household farmers , but there have been very few studies on the impacts of household land management ( land use , land tenure status and land fragmentation ) on soc status and sequestration potential of soils .
china has experienced profound changes in land use , land cover and land management systems over the last 50 years . since the late 1970s ,
farmland management has shifted from collective farming to a household responsibility system , which has resulted in small - scale household farms with intense land fragmentation ( tan et al .
the impacts of farmland management on cropland soil quality and productivity have been investigated , examining the impacts of land fragmentation ( tan et al .
2003 ) and/or household behaviours related to technology transfer ( zhang & xu 1996 ; ou - yang et al .
2003 ) . with growing concerns for climate change mitigation in agriculture , sequestration of soc in croplands may play a key role in off - setting the increasing ghg emissions in china
changes in soc storage and dynamics of china 's croplands have been increasingly studied under land use change ( song et al .
2005 ; hou et al .
2007 ; li et al .
2007 ) ,
fertilization practice ( wang et al .
2010 ) , cropping and rotation ( xu et al .
2006 ) and tillage practice ( wang et al .
2009 ) . while studies have indicated a significant biophysical potential for soc sequestration in china 's croplands ( lal 2002 , 2004b ; pan et al .
2010 ) , there has been little information on how , and to what extent , household farm management behaviours impact on soc storage and the c sequestration capacity of china 's croplands .
this information gap limits the ability to assess the realizable contribution of soc sequestration to the mitigation of ghgs in china in the future and weakens the development of climate policy for cropland management ( pan 2009 ) .
the purpose of the present paper is to quantify the variation of soc storage in household farms under different land management settings , with respect to the land tenure status , household farm size and land use , using the dataset from a farm inventory conducted in 2003 . from this analysis ,
the aim is to provide information for policy - making for improving farm management to enhance c sequestration and ghg mitigation in china 's agriculture .
an inventory of crop production and soil management at household farm scale was conducted in banqiao village in honghu township , yujiang county ( 28042837n , 1164111709e , 4550 m asl ) , jiangxi province , china ( fig .
this village is considered representative of the typical rural conditions that can be found in northeast jiangxi province and in the much larger hilly area of southeast china with rice - based production systems ( kuiper et al .
the local climate is governed by a subtropical monsoon , with a mean annual temperature of 172181c and annual precipitation of 17001800 mm , with 07 of that occurring in late april
early july for the last two decades ( jiangxi bureau of land management jblm 1991 ) .
the croplands are mainly derived from red soils , which are extensive in this area .
the soil type in the rice paddies was classified as hydro - agric stagnic anthrosols , according to chinese soil taxonomy ( gong et al .
2007 ) , and as typic paleudults according to soil taxonomy ( sss - usda 1999 ) , whereas those for dry croplands were inceptic paleudults ( sss - usda 1999 ) .
poor soil fertility and crop productivity in this area are apparent due to degradation of the cultivated red - earth - derived soils ( zhao 2002 ) .
fig .
1sketch map of jiangxi province of china showing the location of the surveyed village in yellow solid circle .
sketch map of jiangxi province of china showing the location of the surveyed village in yellow solid circle .
the village surveyed was among those of poor agricultural productivity within the least developed economy in jiangxi , china during 197090s , and most of the farmers lived on agricultural output before the survey was conducted in 2003 ( tan et al .
the village had 220 households and a population of 900 in 2002 , divided into four hamlets with 1133 ha of farmland in total , giving a land - use density of 015 ha per capita .
of the total farmland area , irrigated rice paddies accounted for 823 ha mostly with double rice cropping , and a further 311 ha farmland was dry croplands , mainly peanuts and vegetables plus citrus trees .
the land tenure system shifted to the household responsibility system in 1984 ; farmlands were allocated to households according to family size and labour force , and by considering soil quality and distance from the village . for the survey ,
15 households , managing a total of 105 plots , were randomly selected from a single hamlet within the whole area of 1133 ha farmland affiliated with the village ( banqiao ) .
plot size , history of cultivation , yield and management ( including land tenure status , crop rotation system , fertilization , straw return and soil fertility condition ) for the past 5 years were recorded for each of the 105 plots .
rice paddies in terraces with pump irrigation have been developed since the 1960s , whereas dry land crops have persisted in the village for over 100 years .
the 105 plots sampled were distributed randomly among the total farmland affiliated with the village .
topsoil samples of all the plots associated with the 15 households surveyed were collected at a depth of 0150 mm using a soil core sampler ( eijkelkamp , the netherlands ) in late february 2003 , when no crop growth was present .
a composite sample was formed with three randomly selected subsamples for each plot , mixed after sampling in the field .
each sample was placed in a stainless - steel box and shipped to the laboratory within 3 days of sampling .
the basic soil physical and chemical properties of the soils tested are summarized in table 1 .
table 1basic soil properties ( means.d . ) of the topsoil samples ( 0150 mm ) ( n=105 ) basic soil properties ( means.d . ) of the topsoil samples ( 0150 mm ) ( n=105 ) soil total organic c and nitrogen ( n ) were measured using an elementar vario max cns analyser ( elementar gmbh , germany ) .
the determinations of soil ph ( h2o ) , available phosphorus and potassium and clay content were conducted following the protocol described by lu ( 2000 ) .
the topsoil soc values of croplands from the village surveyed and measured during the second national soil survey , completed in 1985 , were retrieved from the records available in the local soil survey service of yujiang county .
statistical differences in soc between the land use types and land tenure status were tested with analysis of variance ( anova ) using the spss 11.0 statistical package ( spss 2001 ) .
the basic information on the land tenure and land use status of the household farmland is presented in table 2 .
the mean size of the households surveyed was 45 persons with , on average , 067 ha farmland per household .
the land use density of 015 ha per capita is larger than the mean for china as a whole , which was 009 ha per capita in 2004 , reported by the ministry of land and resources through a national land use change survey ( nacoc & fasc 1999 ) . of the total 105 plots surveyed ,
83 were used for rice paddies and 22 were for dry croplands , reflecting the rice - dominated agriculture in south china .
rice cultivation occupied 09 of the total cultivated area in jiangxi in the late 1980s ( jblm 1991 ) and 085 of the total of the local county of yujiang ( ssoyc 1986 ) .
reform of the land tenure system was finalized in 1985 ; most of the plots surveyed were used by households who contracted them directly from the village collective .
however , 21 plots were leased from other households within the same village for at least one crop growing season .
table 2general status of land management and farmland plot size ( means.d . ) of the 15 households surveyed in banqiao village general status of land management and farmland plot size ( means.d . ) of the 15 households surveyed in banqiao village as shown in fig .
2 , the plot size of the household farmlands followed a skewed distribution and ranged from 001 to 063 ha , with the majority of plots being small .
the mean plot size of 011 ha for the household farmland plots surveyed was close to that of china as a whole ( 009 ha ) . a large degree of land fragmentation occurred in these household farmlands ; 47 plots had a mean size of 005 ha compared to a mean size of 022 ha in the 58 plots with a plot size more than 01 ha ( table 2 ) .
furthermore , a significantly higher degree of land fragmentation was associated with leased plots and with dry croplands ( table 2 ) .
these features of the land tenure system make it more difficult to enhance productivity and , in turn , soc accumulation of the croplands .
observation frequency of size distribution of the 105 plots occupied by the households surveyed . following a quasi - normal distribution ( fig .
3 ) , which was statistically tested , the soc contents of the 105 plots ranged from 17 to 252 g / kg with a mean ( s.d . ) of 1361 g / kg .
thus , the variability of plot soc is significantly smaller than the variability of plot area itself .
the variation of topsoil soc content under different status of land tenure , land fragmentation and land use is presented in fig .
the majority of the 105 plots were rice paddies with a mean soc content of 1395 g / kg , being significantly higher ( 70% higher , on average ) than the dry croplands .
this is in general agreement with previous findings of much higher soc contents in rice paddy soil than in dry croplands ( pan et al .
soc contents were much higher in contracted than in leased plots , with the mean value of the former almost double the latter .
the effect of land fragmentation on soc content was observed to be significantly smaller , as plots < 01 ha had significantly lower soc levels ( by up to 20% on average ) than those > 01 ha .
land fragmentation associated with farm size had an impact on soc . for households with smaller numbers of plots ,
those managed by households with a greater total area of farmland showed significantly higher soc contents ( by 34 g / kg on average ) than those managed by households with smaller total area of farmland in 2003 ( table 3 ) . compared to the original soc level in 1985 ,
when the household responsibility system was newly implemented , soc accumulation of 612 g / kg was found in plots managed by households with > 07 ha total farmland area , whereas it was 1 g / kg for plots managed by households with < 07 ha total farmland .
plots managed by households with larger farm size and smaller numbers of plots were significantly richer in soc than those managed by households with larger numbers of plots .
since the implementation of the household responsibility system , soc has increased in the larger household farmlands , by over 6 g / kg on average , compared to no significant increase in those with smaller total farmland area .
the findings suggest a large interactive impact of land tenure system and land use on both soc storage and soc dynamics of household farmlands .
fig .
3frequency distribution of topsoil organic c content of the 105 plots occupied by the households surveyed .
4variation of topsoil organic carbon content with land management , land use and fragmentation of the 105 plots surveyed .
different capital letters above the bar indicate significant difference at p<005 between types in a same block .
table 3changes in farmland topsoil soc ( g / kg ; meansd ) with number and area of plots affiliated to the 15 surveyed households frequency distribution of topsoil organic c content of the 105 plots occupied by the households surveyed .
variation of topsoil organic carbon content with land management , land use and fragmentation of the 105 plots surveyed .
different capital letters above the bar indicate significant difference at p<005 between types in a same block .
changes in farmland topsoil soc ( g / kg ; meansd ) with number and area of plots affiliated to the 15 surveyed households the area is characterized as being of low productivity for rice due to the poor soil quality and low - nutrient pools , as the soils on the red soil terrace have suffered from acidity and severe soil erosion ( zhao 2002 ) .
previously , in order to meet the demand for higher cereal production , rice was double- or triple - cropped , and without conservation practices this has largely exhausted the soil fertility . improved agricultural management in the area
would entail a cropping system with straw return and green manure cultivation ( tan et al .
as seen in table 4 , most of the plots had been cultivated with double or triple cropping of rice , with very little green manure cultivation or straw return . for dry croplands , there was a single cropping of peanuts and fruit trees with poor soil conditions , with double and triple cropping predominantly used for rice rice and rice rice
while the topsoil soc under single cropping of dry crops tended to be much lower than the double and triple cropping with rice , there was only a small difference in soc contents between the double- and triple - cropped plots .
cultivation of green manure crops , mainly alfalfa in this region , yielded significantly higher soc contents than the non - green manure cultivated plots ( 33 g / kg more on average ) . however , straw return resulted in an increase in soc by c. 5 g / kg compared to plots without straw return .
comparatively , the effect of management practices on topsoil soc was greatest with straw return , followed by green manure cultivation and least with rice cropping intensity .
table 4topsoil soc content ( means.d . ) of plots with different agricultural management practices topsoil soc content ( means.d . ) of plots with different agricultural management practices
the mean ( s.d . ) topsoil soc level of the village farmlands , analysed and reported for the second national soil survey of jiangxi in 1985 for the same soil sampling depth , were 1248 and 729 g / kg for rice paddies and dry croplands , respectively ( q. zhang , personal communication ) .
however , in the present study , the household farmlands surveyed in 2003 had topsoil soc contents of 1457 and 852 g / kg for rice paddies and dry croplands , respectively ( fig .
the changes in deep profile with management practices seemed insignificant ( zhou et al .
there was an increase in topsoil soc at 23 and 12 g / kg of the surveyed plots , although the current level was lower than the provincial mean of 1762 and 944 g / kg reported by huang ( 1999 ) for rice paddies and dry croplands , respectively .
the general trend of soc increase in the present croplands by plot survey is in accordance with previous findings at the county level in yixing ( zhang et al .
2007 ) from the tai lake region , for jiangsu province level ( liao et al .
2009 ) and across china 's croplands ( pan et al .
greater soc enhancement in rice paddies than in dry croplands has also been reported at the regional scale ( hou et al .
2007 ; xu et al .
2009 , 2010 ) for generally higher inputs and lower decomposition under submerged conditions in rice paddies than in dry croplands
. the mean rate of increase for rice paddies and dry croplands of 014 and 007 g / kg / yr , respectively , are somewhat higher than those of 011 and 006 g / kg / yr across china 's croplands ( pan et al .
this could be , in part , attributed to the chemical protection of soc by binding to oxyhydrates in the soils derived from red earth ( zhou et al .
zhou et al . ( 2009b ) reported a faster and greater c sequestration in a red - soil - derived paddy under long - term experiments when comparing with other two rice paddy soils lower in free oxyhydrates .
however , higher increases have been reported for croplands from adjacent areas : xu et al .
( 2009 ) showed mean increases of 025 and 009 g / kg / yr in rice paddies and dry croplands , respectively , at the county level in guichi , anhui province , with predominantly lowland croplands derived mainly from wetlands along the yangtze river . in a previous study ,
an increase in soc of 028 g / kg / yr was observed during 19852002 in the rice paddies in yixing county , jiangsu province ( zhang et al .
2004 ) , where larger - scale farms had been developed , and the cropping system of rice rape and rice
winter fallow had been dominant since the late 1980s , with rapid industrialization ( ma & liu 2009 ) .
this , together with variation of soc contents with the different farm scales and plot sizes may reflect the impacts of land tenure arrangements and land fragmentation on cropland soc storage and enhancement .
3 , tables 3 and 4 ) of plot topsoil soc level reflected the interactive effects of agricultural management practices and household land management .
agricultural management practices had differing effects on topsoil soc ; the increases were : 293% for green manure cultivation ; 416% for straw return ; 438% for rice cropping intensity ( 1654 g / kg under double rice to 1149 g / kg under single rice ) ; and 549% for total cropping intensity ( 1557 g / kg under triple cropping to 969
in contrast , aspects of household land management showed land fragmentation to have an effect on topsoil soc : plots over 0.1 ha had 193% more soc than plots smaller than 01 ha ; as did land use type : rice paddies had 722% more soc than dry croplands ; and land tenure status showed land managed under direct contract had 965% more soc than leased land ( fig .
4 ) . while the effects of land fragmentation on crop productivity and resource use efficiency have been widely debated ( wan & cheng 1996 ; su & wang 2002 ; tan et al .
2006 , 2008 ) , fragmentation was not a critical factor in the present study .
a large reduction in soc did occur when paddy was converted to dry cropland , which has also been observed elsewhere ( hou et al .
2007 ;
. while agricultural management practices had direct effects on soc through changes in organic matter input from the crop ( especially with straw return and green manure cultivation ) , land tenure status had indirect effects through the above - mentioned organic matter input .
conservation practices have largely been neglected by the household farmers over the last decade ( wan & cheng 1996 , 2001 , 2005 ; yu et al .
the land managers were more concerned with obtaining adequate yield rather than ensuring soil quality and fertility ( yu et al .
in addition , most of the fragmented , small cropland plots in the village were the dry croplands on degraded red earth and with less accessible irrigation .
due to the relatively high cost of managing these fragmented lands of poor productivity , there is no incentive for the farmers to manage the land carefully ( tan et al .
in fact , the fragmented , poor croplands some distance away from the village were those usually put for lease and used for dry crop production ( tan et al .
2008 ) , which has resulted in reduced storage of soc overall . to examine the effect of good v. poor household and agricultural management ,
scenario i consisted of 19 plots under double - cropped rice with straw return or green manure application , under direct contract and > 01 ha .
scenario ii comprised 15 plots of dry croplands without green manure or straw return , under lease and < 01 ha . land tenure and the agricultural management practices of croplands can have large synergistic effects on soc storage .
storage of topsoil soc was much smaller ( 7210 g / kg on average ) in poorly managed plots ( scenario ii ) compared to those under the beneficial land use and management of rice paddies of scenario i ( 1853 g / kg on average ) .
this suggests a large gap in the soc stock of farmlands due to household land management , which may thus have great impacts on soc levels in china 's croplands superimposing the agricultural management effects at farm scale .
concern has been expressed previously that china 's agriculture has been constrained by both the shortage of arable land and land fragmentation ( wan & cheng 1996 ; su & wang 2002 ; wu et al .
2005 ) .
the need for improved management to enhance cereal production and increase agricultural production efficiency has often been highlighted ( zhang & xu 1996 ; luo 2008 ; lu & zhang 2008 ; wu et al .
management for enhancing crop productivity will benefit soil c sequestration ( lal 2004a ) , and organic c accumulation favours crop productivity in china 's croplands , especially for rice paddies ( pan & zhao 2005 ; pan et al .
thus , developing a sound household land management system , in addition to good agricultural management practices , is necessary to help ensure cereal production , and c sequestration for mitigating ghg emissions in china .
therefore , incentives for encouraging good household farm management practices at the appropriate scale should be taken into account in china 's national action plan for climate mitigation .
more broadly , the land tenure and household land management system seen in this region is very common throughout china , and has a number of similarities with systems in many other developing countries .
the village studied was among those living on agricultural production in a rural area far from the cities ( at least 150 km from the nearest city of yingtang , jiangxi as shown in fig .
the size and total area , as well as the crop production , were typical for the south china 's rural area ; the mean size of total farmlands per household was < 1 ha .
crop production was mainly rice , plus some dry crops in the rolling uplands with infertile soils .
most of the south asian and african countries previously had such landform and agriculture as well as land tenure systems in rural areas ( mcpherson 1982 ; otsuka et al .
2001 ;
the present case study provides rare quantitative information linking limits in the efficacy in soil c storage with potential barriers to implementation , and may serve as a template for investigating barriers to implementation of climate mitigation practices in agriculture elsewhere in the developing world . | summaryan inventory of topsoil soil organic carbon ( soc ) content in household farms was performed in a village from a red earth region in jiangxi province , china in 2003 . in this region ,
the farmland managed by each household is fragmented , consisting of several plots of land that are not necessarily adjacent to each other .
a statistical analysis of soc variation with land use and household management type , and with crop management practices was conducted .
plot size ranged from 0007 to 0630 ha with a mean of 01 ha , and soc content ranged from 172 to 252 g / kg , varying widely with a variety of land management and agricultural practices , arising from individual household behaviours .
the mean soc content in plot size < 01 ha was 20% lower than in plot size 01 ha .
soc of dry crop plots was 70% lower than that in rice paddies , and soc of plots contracted from the village was almost double that of plots leased from other householders .
moreover , a 30% increase in soc was observed with green manure cultivation , and a 55% increase under triple cropping .
the difference in soc levels between the least and most favourable cases of household land management and agricultural practice was up to 150% .
the results suggest that policies targeted at crop management alone may not deliver the expected soc benefits if household land management is also not improved . |
a prospective study conducted at our hospital involved 41 pregnant women who bore a total of 41 fetuses and visited our department for level - ii obstetric us .
gestational age ranged from 12 to 37 ( mean , 25 ) weeks : for nine , it was 10 - 20 weeks ; for 23 , 21 - 30 weeks ; and for the remaining nine , more than 30 weeks . among these 41 women ,
the findings of fetal us were normal in 31 and abnormal in the remaining ten .
abnormalities included facial cleft ( 2 ) , echogenic thoracic mass ( 2 ) , diaphragmatic hernia ( 1 ) , omphalocele ( 1 ) , duodenal atresia ( 1 ) , echogenic bowel ( 1 ) and dilatation of the renal pelvis ( 2 ) .
the diagnosis was confirmed at delivery or autopsy in five of these ten patients , two were followed - up for five months , and the remaining three were lost to follow - up . in two fetuses with a dilated renal pelvis , postnatal renal us findings were normal .
all images were obtained using a 2 - 5 mhz convex transducer ( hdi 5000 ; advanced technology laboratories , bothell , wash .
cus was used first , followed , successively , by thi and pihi , and for each technique , standard institutional us protocols were applied and between 20 and 30 images were obtained ; in each instance , the same depth , magnification and focus were used , and gray - scale gain was adjusted to optimize image quality .
all cus , thi , and pihi images obtained were evaluated by two radiologists using a 22k picture archiving and communication system monitor ( general electric medical systems integrated imaging solutions , mt .
one or two representative images for each of six organ systems , namely the brain , spine , heart , abdomen , extremities and face , were selected for review ( table 1 ) .
after reviewing the given standard planes obtained using the three techniques , the overall conspicuity of each organ system was subjectively assigned one of four grades ( 4 - excellent , 3 - good , 2 - fair , or 1- poor ) , depending on the definition of borders and internal structures .
the grades were averaged between the readers , and cus , thi , and pihi were compared . during the review , each reader
in the ten fetuses whose us findings were abnormal , the conspicuity of these anomalies was graded by one radiologist , using the same system .
grades for the three different techniques were then compared . for multiple statistical comparison between the three techniques ,
friedman 's test was used ; a p value of less than 0.05 was considered statistically significant . to establish interobserver agreement , individual data sets from the two readers were compared .
cus , thi and pihi were quantified using their weighted kappa value , with a 95-percent confidence interval .
a comparison of overall conspicuity and definition of the borders and internal structures of the six organ systems , as seen at cus , thi , and pihi , appears in table 2 . for the brain , heart , abdomen and face ,
the overall conspicuity of fetal organs was significantly better at thi and pihi than at cus ( fig .
1 ) ( p < 0.05 ) , though there was no significant difference between thi and pihi . for the spine ( fig .
in two first - trimester fetuses , thi and pihi delineated internal structures better than cus ( fig .
in the ten abnormal fetuses , the abnormality was more clearly delineated at both thi and pihi than at cus ( p < 0.05 ) ( table 3 ) .
this was so , for example , for a body - wall defect and a liver - containing sac in the fetus with omphalocele .
the dilated renal pelvis present in two fetuses was clearly depicted using all three techniques , though soft - tissue contrast was slightly better and the near - field artifact was smaller at thi and pihi than at cus ( fig .
4 ) . in the two fetuses with echogenic thoracic masses , the lesions contrasted better with surrounding normal lung at thi and pihi than at cus .
in the two fetuses with a facial cleft , the lesions were clearer at thi and pihi than at cus .
the overall kappa value was 0.845 ( very good agreement ) , with a 95 percent confidence interval of 0.831 - 0.888 .
for the three techniques , all kappa values calculated ranged between 0.711 and 1.000 ( table 4 ) .
a comparison of overall conspicuity and definition of the borders and internal structures of the six organ systems , as seen at cus , thi , and pihi , appears in table 2 . for the brain , heart , abdomen and face ,
the overall conspicuity of fetal organs was significantly better at thi and pihi than at cus ( fig .
1 ) ( p < 0.05 ) , though there was no significant difference between thi and pihi . for the spine ( fig .
in two first - trimester fetuses , thi and pihi delineated internal structures better than cus ( fig .
in the ten abnormal fetuses , the abnormality was more clearly delineated at both thi and pihi than at cus ( p < 0.05 ) ( table 3 ) .
this was so , for example , for a body - wall defect and a liver - containing sac in the fetus with omphalocele .
the dilated renal pelvis present in two fetuses was clearly depicted using all three techniques , though soft - tissue contrast was slightly better and the near - field artifact was smaller at thi and pihi than at cus ( fig .
4 ) . in the two fetuses with echogenic thoracic masses , the lesions contrasted better with surrounding normal lung at thi and pihi than at cus . in the two fetuses with a facial cleft , the lesions were clearer at thi and pihi than at cus .
the overall kappa value was 0.845 ( very good agreement ) , with a 95 percent confidence interval of 0.831 - 0.888 .
for the three techniques , all kappa values calculated ranged between 0.711 and 1.000 ( table 4 ) .
owing to the presence of a natural sonic window of amniotic fluid , us is the most suitable modality for the diagnosis of fetal abnormalities , and its safety and availability have facilitated its wide use in nearly all pregnancies .
cus is , however , of limited utility for the evaluation of obese patients and those with poly- or oligohydramnios , uterine anomalies , or multiple myomas ( 20 - 21 ) .
thi is a us technique in which the us beamformer transmits at one frequency and the transducer receives at twice that frequency ( harmonic frequency ) . using a filter to process the received signal , only the returning second harmonic signal is used to produce the image .
the use of a narrow us beam width provides better lateral resolution and improved depiction of the details of small anatomic structures ( 24 - 25 ) . additionally , because of the smaller dynamic range involved
, tissue contrast resolution is accentuated , and since nonlinear propagation causing waveform distortion and the diffraction effect is less , thi produces fewer artifacts than cus ( 25 ) .
pihi is a newer technique free of the bandwidth limitation of thi ( 9 ) . by transmitting two mirror - imaged pulses of alternating phases ,
the fundamental signals are cancelled out , generating a pure harmonic signal ( 26 ) , and very high spatial resolution is known to result . while thi uses a narrow receiving filter , pihi uses broader transmitting and receiving bandwidths , and resolution is thus improved ( 24 ) .
the well - known disadvantages of pihi are its slow frame rate and flashing artifacts , though in fetal imaging these do not seem to often cause problems .
soft tissues , including the skin , subcutaneous tissue and musculature , seem to be more conspicuous at thi than at cus . in our study , thi and pihi were superior to cus for the brain , heart , and abdomen , but pihi was better than cus for evaluation of the face .
in contrast , bony structures were not clearly delineated at thi or pihi ; they were more often obscured by clear posterior sonic shadowing of bones than at cus ( fig .
2 ) , a fact which may explain the lower grades of conspicuity seen at thi and pihi of the spine and extremities . for these areas , there were no statistically significant differences between the three techniques .
the conspicuity of all six organ systems and overall image quality were similar at pihi and at thi , though among the three techniques , near - field artifacts in fetal brain beneath the calvarium and in fetal abdomen beneath the abdominal wall were smallest at pihi ( fig .
published reports have not addressed the question of the safety of thi and pihi in fetal imaging . although egerton et al . ( 27 ) reported possible increases in tissue temperature during harmonic imaging , thi and pihi without contrast appear , in theory , to be safe in an ordinary clinical setting .
thi obtains weak harmonic signals generated in the body , and except for the selection of a narrower bandwidth frequency , the transmitted sound beam remains the same as with cus .
pihi uses two sound pulses per scan line , resulting in more sound energy per line ; as the frame rate is halved , however , average sound energy over time is the same . for this reason , both
the thermal and mechanical indices encountered in clinical practice , using either technique , usually remain the same as with cus .
interobserver agreement in our study was very good , with close correlation between the findings of the two reviewers , an overall weighted kappa value of 0.845 , and a 95-percent confidence range of 0.831 - 0.888 .
there was very close agreement ( 0.711 - 1.000 of the weighted kappa value ) between the two observers for both normal and abnormal fetuses , and all six organ systems .
this result demonstrates , we believe , that both thi and pihi are consistently superior to cus and can thus serve as useful tools for imaging of the fetus .
first , since the image characteristics of the three different techniques were recognizably different , the readers were not blinded , during their analysis , to which had been employed .
second , we included in this study only patients who visited our department for level - ii fetal us , and for this reason , the gestational age of the fetuses varied , and was not well controlled .
fetal imaging findings are strongly influenced by gestational age , and our thi and pihi findings may , for this reason , have been affected .
third , only ten abnormal fetuses were studied , and to determine the usefulness of these techniques and the applicability of our findings , further investigation by means of a well - designed prospective study is warranted . in conclusion , both thi and pihi are superior to cus for the evaluation of normal and abnormal fetuses , particularly for assessment of the brain , heart , abdomen , and face . because it provides high tissue contrast resolution and artifacts are fewer , harmonic imaging may be particularly useful in the evaluation of internal fetal organs . | objectiveto determine the usefulness of tissue harmonic imaging ( thi ) and pulse - inversion harmonic imaging ( pihi ) in the evaluation of normal and abnormal fetuses.materials and methodsforty - one pregnant women who bore a total of 31 normal and ten abnormal fetuses underwent conventional ultrasonography ( cus ) , and then thi and pihi . us
images of six organ systems , namely the brain , spine , heart , abdomen , extremities and face were compared between the three techniques in terms of overall conspicuity and the definition of borders and internal structures.resultsfor the brain , heart , abdomen and face , overall conspicuity at thi and pihi was significantly better than at cus ( p < 0.05 ) .
there was , though , no significant difference between thi and pihi . affected organs in abnormal fetuses were more clearly depicted at thi and pihi than at cus.conclusionboth thi and pihi appear to be superior to cus for the evaluation of normal or abnormal structures , particularly the brain , heart , abdomen and face . |
glaucoma is a multifactorial , progressive optic neuropathy characterized by retinal ganglion cell death , the loss of visual field , and excavation of the optic nerve head .
glaucoma , the second leading cause of preventable blindness , is usually caused by the effects of elevated intraocular pressure ( iop ) . as elevated iop is the only treatable risk factor for disease ,
glaucoma surgery is indicated if target pressures are not achieved , or if the glaucomatous optic neuropathy progresses despite maximally tolerated medical and laser therapies.1 currently , trabeculectomy is still the most reliable and most widely performed surgical procedure to lower iop in the vast majority of patients with uncontrolled glaucoma despite maximal medical therapy .
trabeculectomy allows the drainage of aqueous humor from the anterior chamber to the subconjunctival space by creating a half - scleral - thickness limbal fistula .
this procedure reduces iop by a filtering bleb.24 the failure of glaucoma filtration surgery ( gfs ) is usually due to excessive wound healing under the conjunctiva or over the scleral flap causing failure of the filtering blebs .
thus , suppression of the wound healing response is very important in preventing obstruction of the established fistula.5,6 fibroblasts are the primary cell type to play a role in wound healing in the scleral flap region following gfs . in addition ,
mononuclear cells ( mncs ) are well known to be the primary source of fibrogenic and angiogenic cytokines such as transforming growth factor ( tgf ) , fibroblast growth factor ( fgf ) , and platelet - derived growth factor ( pdgf ) , which play an authoritative part in initiating wound healing .
these growth factors cause the most potent influence on fibroblasts , and they increase the activity of fibroblasts and enhance the secretion of extracellular matrix components.57 thus , the suppression of fibroblasts and mncs in the surgical region increases the success of trabeculectomy . currently , there are many anti - fibrotic agents used in the suppression of wound healing after gfs .
mitomycin - c ( mmc ) and fluorouracil are the most used anti - fibrotic agents to increase the success of trabeculectomy .
however , they have serious adverse effects such as corneal endothelial toxicity , bleb leaking , hypotonous maculopathy , blebitis , and endophthalmitis.812 thus , new agents that are more selective and less toxic are needed to suppress wound healing after gfs .
infliximab is a mouse / human chimeric monoclonal igg1 antibody against tumor necrosis factor ( tnf)-. tnf- is a local paracrine and autocrine regulator for low - density leukocyte and endothelial cells .
it stimulates mononuclear phagocytes as well as other cell types that produce various proinflammatory cytokines and chemokines and induces migration of polymorphic nuclear leukocytes .
infliximab binds to the tnf- molecule and reduces lymphocyte migration and production of proinflammatory cytokines , including interleukin ( il)-1 , il-6 , and adhesion molecules .
it is an agent used in the treatment of disorders such as crohn s disease , ulcerative colitis , rheumatoid arthritis , ankylosing spondylitis , and autoimmune ocular inflammatory diseases such as uveitis , scleritis , and behet s disease.1315 it was also recently reported that infliximab is effective in treating choroidal neovascularization and in the prevention of corneal neovascularization.16,17 in recent studies , it was demonstrated that infliximab downregulated expression and levels of the cytokines that play a role in the wound healing response , including tgf- , basic fgf , and pdgf - b.1820 to the best of our knowledge , there is no previous study in the literature on the suppression of wound healing with topical infliximab following gfs . in our study
, we aimed to investigate the effects of infliximab on wound healing in experimental gfs and to compare the anti - fibrotic effects of this agent with those of mmc .
the animals were kept under appropriate dietary conditions in special cages in the experimental research center at frat university , elaz , turkey throughout the study .
the animals were housed in wire - bottomed individual cages at room temperature ( air - conditioned , 22c26c ) on a 12-hour light dark cycle .
all of the rabbits were acclimatized for at least 1 week before the experiments started .
the animals were fed standard rabbit chow , but were given only water 12 hours before surgery .
the study was approved by the institutional animal care and use ethics committee of frat university .
all experiments and the animal - related procedures were performed with strict adherence to the guidelines for animal care and experimentation by the association for research in vision and ophthalmology ( arvo ) as described in their statement for the use of animals in ophthalmic and visual research .
only one eye of the rabbits in the study groups was subjected to standard preparation for surgery , anesthesia , and the surgical technique .
the rabbits were randomly assigned to four groups , each including seven rabbits : control group , sham group , mmc group , and infliximab group .
the rabbits in the control group were not operated on and did not receive any treatment .
the rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days . the rabbits in the mmc treatment group underwent trabeculectomy , and a sponge soaked in 0.4 mg / ml mmc was applied intraoperatively to the scleral surgical site for 3 minutes .
the rabbits in the infliximab treatment group underwent trabeculectomy , and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . a combination of intramuscular 50 mg / kg ketamine hydrochloride ( ketalar ; eczacibasi holding co , istanbul , turkey ) and 6 mg / kg xylazine hydrochloride ( rompun ; bayer ag , leverkusen , germany ) was used in the anesthesia and analgesia . before the operation , a single drop of 0.5% proparacaine hydrochloride ( alcaine ; alcon laboratories , inc .
, fort worth , tx , usa ) was instilled in the eyes to be operated on .
the surgical procedure was performed using an operating microscope ( carl zeiss meditec ag , jena , germany ) a limbal - based trabeculectomy , as described by cairns,21 was performed on the right eyes of all the rabbits in the study groups , except for the control group .
briefly , a lid speculum was placed and a partial - thickness 8 - 0 silk corneal traction suture was applied superiorly to pull down and fix the eyeball .
a peritomy was made 5 mm above the limbus to form a limbus - based conjunctival flap .
a limbus - based rectangular ( 2.5 2.5 mm ) scleral flap was outlined with a steel blade and carefully dissected .
a trephine ( 1.5 mm in diameter ) was then applied to make the entry into the anterior chamber at the surgical limbus , and a peripheral iridectomy was performed .
the site of the peripheral iridectomy led to detection of the location of the bleb in the histopathologic examination .
the scleral flap was closed by suturing with 10 - 0 nylon at two points , and the conjunctival wound was closed by suturing with two 9 - 0 polyglactin sutures .
the rabbits in the sham group received one drop of saline four times a day for 14 days after the surgery . in the mmc group , a sponge soaked in 0.4 mg
/ ml mmc was applied to the scleral surgical site for 3 minutes , after which the application area was thoroughly irrigated with 10 ml of balanced salt solution .
the rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . at the end of the 14th postoperative day ,
the eyes were enucleated , the rabbits were anaesthetized again , and the globes were enucleated together with the conjunctiva , including the bleb , for histologic examination .
single ( right ) eyes of the rabbits in the control group were also prepared for histologic examination .
the operation site of each enucleated eye was dissected as a blockage , which contained the conjunctiva , tenon s capsule , and sclera .
the tissue specimens were fixed in 10% formaldehyde and embedded in paraffin before the sections were cut .
serial sections ( 4 m thick ) were cut , dehydrated , and stained with hematoxylin and eosin stain for light microscopy examination .
the specimens were stained with masson s trichrome , and microscopic analysis was performed using 400 objective of a standard light microscope ( bx50 photomicroscope ; olympus corporation , tokyo , japan ) . using a specific micrometer attached to the microscope , fibroblasts and mncs in ten randomly determined sections ( area of 50 m ) were counted , and the average numbers of cells in these zones were analyzed .
the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation .
the slides were stained with tgf- , fgf- , and pdgf kits ( bioss inc . , woburn , ma , usa ) in an automated immunostainer device ( benchmark xt ; ventana medical systems , inc . , tucson , az , usa ) according to the manufacturer s instructions .
the slides , covered with a special cover substrate , were randomly evaluated under a light microscope ( olympus bx50 photomicroscope ) .
digital photographs of the tissues were taken at 40 magnification using the camera on the microscope .
immunostaining intensities of tgf- , fgf- , and pdgf were determined semiquantitatively as none ( 0 ) , weak ( 1 ) , moderate ( 2 ) , or intense ( 3 ) .
statistical analysis of the study was performed with ibm spss statistics software ( v 21 ; ibm corporation , armonk , ny , usa ) to determine the differences between the study groups . the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation . for multiple comparisons , a one - way analysis of variance test
tgf- , fgf- , and pdgf immunostaining differences were assessed with the kruskal wallis test .
the rabbits were randomly assigned to four groups , each including seven rabbits : control group , sham group , mmc group , and infliximab group .
the rabbits in the control group were not operated on and did not receive any treatment .
the rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days .
the rabbits in the mmc treatment group underwent trabeculectomy , and a sponge soaked in 0.4 mg / ml mmc was applied intraoperatively to the scleral surgical site for 3 minutes .
the rabbits in the infliximab treatment group underwent trabeculectomy , and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery .
a combination of intramuscular 50 mg / kg ketamine hydrochloride ( ketalar ; eczacibasi holding co , istanbul , turkey ) and 6 mg / kg xylazine hydrochloride ( rompun ; bayer ag , leverkusen , germany ) was used in the anesthesia and analgesia . before the operation , a single drop of 0.5% proparacaine hydrochloride ( alcaine ; alcon laboratories , inc .
, fort worth , tx , usa ) was instilled in the eyes to be operated on .
the surgical procedure was performed using an operating microscope ( carl zeiss meditec ag , jena , germany ) a limbal - based trabeculectomy , as described by cairns,21 was performed on the right eyes of all the rabbits in the study groups , except for the control group .
briefly , a lid speculum was placed and a partial - thickness 8 - 0 silk corneal traction suture was applied superiorly to pull down and fix the eyeball .
a peritomy was made 5 mm above the limbus to form a limbus - based conjunctival flap .
a limbus - based rectangular ( 2.5 2.5 mm ) scleral flap was outlined with a steel blade and carefully dissected .
a trephine ( 1.5 mm in diameter ) was then applied to make the entry into the anterior chamber at the surgical limbus , and a peripheral iridectomy was performed .
the site of the peripheral iridectomy led to detection of the location of the bleb in the histopathologic examination .
the scleral flap was closed by suturing with 10 - 0 nylon at two points , and the conjunctival wound was closed by suturing with two 9 - 0 polyglactin sutures .
the rabbits in the sham group received one drop of saline four times a day for 14 days after the surgery . in the mmc group , a sponge soaked in 0.4 mg
/ ml mmc was applied to the scleral surgical site for 3 minutes , after which the application area was thoroughly irrigated with 10 ml of balanced salt solution .
the rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery .
at the end of the 14th postoperative day , the eyes were enucleated , the rabbits were anaesthetized again , and the globes were enucleated together with the conjunctiva , including the bleb , for histologic examination .
single ( right ) eyes of the rabbits in the control group were also prepared for histologic examination .
the operation site of each enucleated eye was dissected as a blockage , which contained the conjunctiva , tenon s capsule , and sclera .
the tissue specimens were fixed in 10% formaldehyde and embedded in paraffin before the sections were cut .
serial sections ( 4 m thick ) were cut , dehydrated , and stained with hematoxylin and eosin stain for light microscopy examination .
the specimens were stained with masson s trichrome , and microscopic analysis was performed using 400 objective of a standard light microscope ( bx50 photomicroscope ; olympus corporation , tokyo , japan ) . using a specific micrometer attached to the microscope ,
fibroblasts and mncs in ten randomly determined sections ( area of 50 m ) were counted , and the average numbers of cells in these zones were analyzed .
the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation .
the slides were stained with tgf- , fgf- , and pdgf kits ( bioss inc . ,
woburn , ma , usa ) in an automated immunostainer device ( benchmark xt ; ventana medical systems , inc . , tucson , az , usa ) according to the manufacturer s instructions .
the slides , covered with a special cover substrate , were randomly evaluated under a light microscope ( olympus bx50 photomicroscope ) .
digital photographs of the tissues were taken at 40 magnification using the camera on the microscope .
immunostaining intensities of tgf- , fgf- , and pdgf were determined semiquantitatively as none ( 0 ) , weak ( 1 ) , moderate ( 2 ) , or intense ( 3 ) .
statistical analysis of the study was performed with ibm spss statistics software ( v 21 ; ibm corporation , armonk , ny , usa ) to determine the differences between the study groups . the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation . for multiple comparisons , a one - way analysis of variance test
the means and standard deviations of the fibroblast and mnc numbers in the study groups are presented in table 1 .
the means and standard deviations of the tgf- , fgf- , and pdgf immunostaining intensities in the study groups are presented in table 2 .
the mean fibroblast and mnc numbers and the mean tgf- , fgf- , and pdgf immunostaining intensities of the sham group were higher than those of the control group ( p<0.01 ) .
the mean fibroblast and mnc numbers and the mean of tgf- , fgf- , and pdgf immunostaining intensities of the mmc and infliximab group were statistically significantly lower than those of the sham group ( p<0.01 ) .
there was no statistically significant difference between the mean mnc and fibroblast numbers and the mean immunostaining intensities of tgf- , fgf- , and pdgf in the mmc and infliximab groups ( p>0.05 ) .
comparative microphotographs showing fibroblast proliferation and mnc infiltration at the bleb site of each of the rabbits in the study groups are shown in figure 1 .
microphotographs of the immunostaining of tgf- , fgf- , and pdgf , of one subject from each study group , are shown in figures 24 , respectively .
antimetabolites such as mmc and fluorouracil have dramatically augmented the success of gfs , especially in patients at higher risk of surgical failure due to excessive wound healing in the surgical region .
however , there is a risk of blindness with antimetabolite use.812 additionally , surgical failure can occur despite the use of these powerful antimetabolites .
thus , there is a need for potent agents that have no severe adverse effects .
fibroblasts , the main cells responsible for excessive wound healing at the scleral flap region following gfs , synthesize collagen , which is the most important component of granulation tissue .
tenon s capsule fibroblast proliferation is stimulated by epidermal growth factor , fgf , tgf- , insulin - like growth factor-1 , pdgf , fibronectin , transferrin , il-1 , and il-6 .
tgf- is the most potent stimulator of fibroblast activity.57,22,23 additionally , after conjunctival and scleral surgery , pdgf is released from platelets , which promotes cell migration into the wound site and increases fibroblast proliferation.6,7,2226 thus , the inhibition of fibroblast function after gfs is an important factor in the wound healing process and for the outcome of gfs . in our study ,
it is possible that infliximab inhibits fibroblast proliferation and migration by toxic effect or by reducing tgf- , fgf- , and pdgf , as seen in our study .
mncs are cells coming to the surgical wound region subsequent to polymorph nuclear leukocytes . the chemotaxis of mncs is increased with fibronectin , growth factors , and the complement system .
macrophages are mncs derived from monocytes and they secrete various factors that stimulate migration and proliferation of the fibroblasts .
they promote early wound healing and play a crucial role in tissue remodeling in the later phases of wound healing.5,22,27,28 macrophages are thought to be the principal source of fibrogenic and angiogenic cytokines such as pdgf and tgf-.29 it has been reported that the conjunctiva of patients whose gfs failed contain more macrophages .
mncs also release enzymes that augment tissue degradation , generate chemotactic factors that recruit inflammatory cells , and interact with lymphocytes .
they produce cytokines and cause the accumulation and proliferation of fibroblasts and endothelial cells , with subsequent fibrosis and angiogenesis.5,22,27,28 thus , it can be put forward that a decreased number of macrophages parallels a suppressed wound healing reaction and surgical success . in our study , mnc infiltration in the bleb region was significantly suppressed in the mmc and infliximab groups ; however , the differences between these two groups were not statistically significant .
the reduction in the number of mncs in both treatment groups was presumably the effect of decreased growth factor levels .
tgf- is produced by macrophages , lymphocytes , platelets , and fibroblasts , and it is one of the most important profibrogenic cytokines in the conjunctival wound healing process , causing the proliferation and migration of human tenon s fibroblasts and promoting collagen production.23,3034 tgf- and pdgf have the strongest influence on wound healing , as these growth factors increase fibroblast activity.35,36 reduced levels of tgf- have been correlated with the retardation of the wound healing process in burn injury and with non - scarring fetal skin repair.37,38 similarly , decreased levels of pdgf have been observed in non - healing wounds in diabetic rats.38 on the other hand , elevated levels of tgf- are associated with increased fibrosis in several organs , including the heart , liver , kidney , lung , skin , and bone marrow.3942 tgf- is considered to inhibit the growth of leukocytes and to stimulate the production of fibroblasts.43,44 tgf- enhances resistance of the wound site to tension in corneal incisions in rabbits by enhancing the number of fibroblasts through chemotaxis , reducing the synthesis of matrix metalloprotease , and enhancing the synthesis of metalloprotease tissue inhibitor.42 in mouse models of conjunctival scarring , it has been reported that tgf-2 applied to mouse conjunctiva following mmc application significantly reversed the anti - scarring effects of mmc.31,32 inhibiting tgf- might be an effective means of preventing postoperative scarring after trabeculectomy . in our study ,
mmc and infliximab similarly reduced the immunostaining density of tgf- at the scleral flap region .
although the exact mechanisms underlying this response are not known , it might be that exposure to mmc reduces human tenon s fibroblasts , which are one of the main producers of tgf-. fgf- secreted by macrophages and endothelial cells stimulates fibroblast migration and proliferation .
tenon s capsule fibroblasts exposed to mmc have been reported to exhibit a significant decrease in the number of fgf- receptors.4044 in our study , mmc and infliximab equally decreased the immunostaining intensity of fgf- in the bleb region . as both drugs strongly suppressed fibroblast proliferation and reduced fgf- ,
pdgf also plays a very important role in wound healing by promoting the proliferation and migration of fibroblasts.4044 studies by denk et al strongly support the hypothesis that pdgf isoforms are major stimulators of proliferation of tenon s capsule fibroblasts after gfs , while tgf- isoforms are essential for the transformation of tenon s capsule fibroblasts into myofibroblasts.25,44 in another study , knorr et al demonstrated that pdgf - ab and -bb isoforms are potent stimulators of the proliferation of human tenon s capsule fibroblasts , suggesting that they play an important role in the wound healing response after gfs.26 pdgf is normally present in trace amounts in the aqueous humor and increases the proliferative response of fibroblasts to tgf-.44 pdgf is also a chemoattractant for trabecular meshwork cells . in turn
, tgf- regulates tissue growth through pdgf protein and receptor expression in keratocytes , although the exact mechanism by which this takes place is unknown . in other connective tissues ,
low tgf- concentrations promote pdgf production , whereas , at high tgf- concentrations , the pdgf receptor expression is decreased.4045 in our study , mmc and infliximab equally decreased the immunostaining intensity of pdgf in the scleral flap region .
mmc is an antimetabolite and antitumor agent that is isolated from streptomyces caespitosus and which decreases fibroblast proliferation in both the subconjunctival space and tenon s capsule .
it is widely used in trabeculectomy to prevent excessive postoperative wound healing in the surgical site in cases at risk of surgical failure , due to its anti - fibrotic and anti - scarring properties .
yamamoto et al reported that mmc inhibited the proliferation and motility of cultured rabbit subconjunctival fibroblasts and that its inhibitor effect depends on dose and time of exposure to the drug.46 another experimental study demonstrated that histologic examination of eyes with mmc - treated trabeculectomy revealed hypocellular and well - formed bleb cavities , and that a single intraoperative application of mmc had a marked effect on postoperative wound healing after filtration surgery in monkeys.47 however , topically applied mmc has severe ocular complications , such as necrosis in the cornea and conjunctiva , scleral perforation , choroidal detachment , epithelial downgrowth , filtration bleb failure , and endophthalmitis.1013 although mmc is widely used in gfs with high success rates , it is not possible to use it in every case that has a high risk of failure .
in addition , mmc is one of the ten most carcinogenic substances to humans.48 infliximab is a human murine monoclonal antibody that can bind to both soluble and transmembranous forms of tnf- with high affinity .
tnf- is a local paracrine and autocrine regulator for small - density leukocyte and endothelial cells .
it stimulates mononuclear phagocytes as well as other cell types that produce il-1 , il-6 , and chemokines , and induces migration of polymorphic nuclear leukocytes .
tnf- has a synergistic effect with il-6 and plays a crucial role in the development of inflammation in uveitis . furthermore
, it contributes to the pathogenesis of uveoretinitis associated with behet s disease.14,15 currently , there is increasing evidence that tnf- is important in the initiation and perpetuation of the fibrotic process.4954 in a recent study , sullivan et al demonstrated that tnf- induces tgf-1 expression in lung fibroblasts.49 battegay et al reported that tnf- stimulates dna synthesis and proliferation of cultured human fibroblasts and that there is a strong relationship between secretion of pdgf and tnf- stimulation.50 therefore , we postulate that tnf- may play a significant role in regulating the expression of the cytokines that themselves play an important role in wound healing after gfs , such as tgf- , fgf , and pdgf .
recently , infliximab has been used as a novel immunomodulatory agent , particularly in uveitis refractory to treatment , panuveitis associated with behet s disease , rheumatoid arthritis , and crohn s disease.55 infliximab was shown to inhibit the functional activity of tnf- in several in vitro studies.56,57 it has been suggested that infliximab exerts its clinical effects in patients with uveitis through various mechanisms , which include reducing serum levels of inflammatory mediators by blocking tnf- , preventing lymphocyte migration , and decreasing levels of vascular endothelial growth factor.58 binding of infliximab to soluble tnf- brings about a decrease in bioactivity , while its binding to membrane - bound tnf- results in cytotoxicity through complement - dependent or antibody - dependent cell - mediated mechanisms . nevertheless , it is not known how infliximab acts in in vivo models .
secondary to the suppression of tnf- , infliximab lowers serum levels of inflammatory mediators , including il-1 and il-6 and vascular growth factors .
in addition , it reduces lymphocyte migration.59 in our study , we observed that topically administered 10 mg / ml infliximab following gfs significantly suppressed fibroblast proliferation and mnc infiltration compared to the sham group , and that it was as effective and strong as mmc .
it was also observed that the tgf- , fgf- , and pdgf immunostaining intensities of the infliximab group were significantly lower than those of the sham group .
it is possible that infliximab acts via the inhibition of fibroblast proliferation and migration by toxic effect or by reducing tgf- , fgf- , and pdgf , as seen in our study . as far as we know ,
following a search of the pubmed database , there is no previous study concerning the use of infliximab in an experimental gfs model .
we consider that infliximab might reduce fibroblast infiltration via the induction of fibroblast apoptosis or suppression of the effects of tgf- , fgf- , and pdgf , as seen in the present study .
the suppressive effect of infliximab on the number of mncs might be related to its cycle arrest effect , apoptosis , or the inhibition of mnc chemotaxis via suppression of the effects of the abovementioned cytokines .
the small number of experimental animals used can be regarded as a main limitation of our study . in this study
, we used a previously reported experimental dosage and administration rate of infliximab.16,59 we did not observe any corneal or conjunctival toxicity in the histologic sections .
taking into consideration the toxic effects of mmc in gfs and the strongly anti - fibroblastic effects of infliximab as well as mmc on conjunctival wound healing , infliximab can be considered a potential alternative agent for the modulation of wound healing after gfs . however , further in vivo and in vitro studies are needed to evaluate its use with gfs . | backgroundthe purpose of this work was to look into the effects of infliximab on wound healing in experimental glaucoma filtration surgery and to compare the antifibrotic effects of this agent to that of mitomycin - c ( mmc).methodstwenty - eight male new zealand white rabbits were randomly assigned to four groups , each including seven rabbits : control group , sham group , mmc group , and infliximab group .
the rabbits in the control group were not operated on and did not receive any treatment .
the rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days . the rabbits in the mmc treatment group underwent trabeculectomy , and a sponge soaked in 0.4 mg / ml mmc was applied intraoperatively to the scleral surgical site for three minutes .
the rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . on day 14 of the experiment , the operated and control eyes were enucleated and histologically and immunohistochemically analyzed.resultsthe mean fibroblast and mononuclear cell ( mnc ) numbers and the mean immunostaining intensities of transforming growth factor- ( tgf- ) , fibroblast growth factor- ( fgf- ) , and platelet - derived growth factor ( pdgf ) in the sham group were higher than those of the control group ( p<0.01 ) .
the mean fibroblast and mnc numbers and the mean immunostaining intensities of tgf- , fgf- , and pdgf in the mmc and infliximab groups were statistically significantly lower than those of the sham group ( p<0.01 ) .
the mean fibroblast and mnc numbers and the mean tgf- , fgf- , and pdgf immunostaining intensities of the mmc and infliximab groups were similar ( p>0.05).conclusionour study suggests that topical infliximab effectively suppresses the subconjunctival wound healing response after experimental glaucoma filtration surgery , reducing the mnc and fibroblast numbers and immunostaining intensities of tgf- , fgf- , and pdgf . |
it has been well known that about half of the patients with lung cancer have metastases at the time of initial diagnosis .
the common sites of distant metastases are the bone , liver , adrenal glands , and lymph nodes .
the clinical symptoms of the metastases of bone , liver , and lymph nodes usually occur relatively earlier .
therefore , these metastases are often diagnosed at the same time the diagnosis of lung cancer was made . however , in the case of metastases to the kidneys , adrenal glands , gastrointestinal tract , and skin , the clinical symptoms of metastasis do not appear early and are usually found at postmortem examination . in particular ,
the intestinal metastases of primary lung cancer are hardly diagnosed until the development of intestinal obstruction , perforation or hemorrhage ; these cases usually show high mortality rate on operation . since morgan s first report in 1961 of small intestinal metastasis from lung cancer ,
27 cases with the clinical symptoms of perforation , obstruction and/or hemorrhage have been reported . among them , only 1 case was intestinal hemorrhage .
we here present a case of intestinal hemorrhage that progressed to intestinal perforation and caused by small intestinal metastasis of carcinoma of the lung , large cell type .
a 31-year - old korean male patient was transferred to our hospital from a local hospital because of persistent melena of undetermined origin .
four months ago , the patient visited the hospital with complaints of chest tightness and productive cough .
an x - ray film of the chest at that time showed a patch infiltration in the right lower lung field .
studies of the sputum for acid fast bacilli were negative but anti - tuberculosis medications were started under the impression of pulmonary tuberculosis .
three months later , he was admitted to the same hospital because of melena and underwent extensive work - ups including gastroscopic examination , barium studies of the colon and small bowels , and a gastric mucosa scan for meckel s diverticulum , but no bleeding focus was found . during the stay in that hospital ,
he did fairly well until 10 days prior to this admission when recurrent melena developed .
additional studies including 99mtc - sulfur colloid scintigraphy were carried out , but no bleeding focus was found . on admission , the patient appeared pale and acutely ill .
the blood pressure was 130/70 mm / hg , pulse rate 120/min , respiratory rate 20/min , and the temperature 36.5c .
the urea nitrogen was 22.4 mg / dl , the creatinine 1.5 mg / dl , the protein 2.5 g / dl ( the albumin 1.3 g / dl and the globulin 1.2 g / dl ) , the bilirubin 0.4 mg / dl , the alkaline phosphatase 53 iu , the aspartate aminotransferase ( ast ) 10 iu , and the alanine aminotransferase ( alt ) 10 iu .
chest x - ray film revealed an ill defined dense infiltrate with air bronchogram in the right lower lung field ( fig .
however , a large amount of hematochezia persisted even after the operation and signs of serious disseminated intravascular coagulopathy ( dic ) follwed . on the 10th hospital day while the dic improved ,
on operation , multiple polypoid masses of various sizes were palpated intraluminally in the jejunum from 10 cm below the treitz ligament to the 120 cm distally .
a large perforation of the jejunum ( 8 cm in diameter ) probably caused by necrosis of the bowel wall was found on the antemesenteric border of the jejunum 2025 cm below the treitz ligament .
multiple mass lesions including hematomes were also found in the mesenteric root of the small intestine . small bowel resection and end - to - end anastomosis were performed . on section , 36 polypoid masses were scattered grossly along the full length of the resected jejunum .
the cells in the polypoid mass lesion were consistent with large round cells of undetermined origin .
bronchoscopic examination , which was done on the 10th postoperative day , revealed an obstructing round mass at the entrance of the right main bronchus ( fig . 5 ) .
histologically , this lesion was diagnosed as large cell carcinoma of the lung ( fig .
it has been reported that 41% of all patients with lung cancer are found to have evidence of distant metastases at the time of initial diagnosis , but the clinical symptoms of the metastases to the gastrointestinal tract are rarely present .
the distant metastases of lung cancer are usually found at the lymph nodes , brain , bone , adrenal glands , and liver through lymphatic spread .
mcneill et al . reported that primary lung cancer metastases to the small intestine was mainly from large - cell type carcinoma as our case .
the peak incidence was in the sixth and seventh decade , similar to those of patients with primary lung cancer .
most of the metastatic small intestinal tumors originated from the cervix , skin , ovaries , and kidneys .
the clinical symptoms were usually those of bowel obstruction , perforation or rarely hemorrhage . by reviewing the literature of the 27 cases reported , 18 had intestinal perforation , 8 had obstruction and only 1 had hemorrhage(table 1 ) .
postmortem examinations of 431 lung cancer patients studied over an 11-year period(1 ) showed that 46 patients ( 11% ) had metastases to the small intestine . among the 46 patients who received exploratory laparotomy with the symptoms and signs of intestinal obstruction or perforation ,
all of these 6 patients died within 16 weeks after the operation . on the other hand , according to the report of arianha et al .
, 10% of patients with suspected metastasis were found to have benign lesions as the cause of small bowel obstruction .
also , in the other 2 reports , 25% and 38% of patients with a history of cancer who underwent exploratory laparotomy because of intestinal obstruction were found to have either a benign disease or a new primary cancer as the cause of the obstruction . according to the report by woods
iv et al , 8 out of 13 patients who underwent emergency exploratory laparotomy experienced successful palliation by surgical intervention .
these data suggested that the cancer patients with intestinal obstrcution who do not have a proven abdominal cancer should undergo exploratory laparotomy .
report a case of perforation caused by metastatic cancer after chemotherapy and they suggested that there might be a close relationship between the chemotherapy and perforation .
inalsingh et al . also explained that local factors , such as trauma and/or irradiation , which could influence the immune response were related to the metastases . among 17 cases with intestinal perforation , 7 presented with perfortion after chemotherapy and radiotherapy . in conclusion , with increasing use of chemotherapy and radiotherapy for the treatment of cancer , the possibility of perforation of the small intestine from the metastatic tumor should be considered more carefully than before . | although intestinal metastases from lung cancer are not rare at postmortem studies , the development of clinically significant symptoms from the gastrointestinal metastases is very unusual .
we report a case of small intestinal hemorrhage leading to intestinal perforation secondary to metastases from a large cell carcinoma of the lung in a 31-year - old man along with a review of the literature . |
primary aldosteronism ( pa ) has been traditionally considered a rare cause of hypertension ( 1 ) .
however , recent studies have shown a higher prevalence , ranging between 5% - 20 % among hypertensive patients ( 2 ) .
moreover , recent studies have demonstrated a higher frequency of cardiovascular complications in pa compared to patients with essential hypertension ( 3 , 4 ) .
endothelial dysfunction could play a role in the deleterious cardiovascular effects of aldosterone , as it predicts long - term atherosclerotic disease progression and the risk of cardiovascular events ( 5 , 6 ) .
asymmetric dimethylarginine ( adma ) is one of the most widely used and validated markers for evaluating the endothelial function .
elevated plasma adma has been identified as a predictor of acute coronary events and an independent risk factor for all - cause and cardiovascular mortality ( 8 , 9 ) .
several studies have reported elevated amda concentration in patients with different cardiovascular and metabolic disorders as essential hypertension ( 10 ) , diabetes mellitus ( 11 ) , hypercholesterolemia ( 12 ) , coronary artery disease ( 13 ) , but reports on adma levels in endocrine hypertension are scarce . increased plasma concentrations of adma could contribute to endothelial dysfunction , increased atherogenesis and elevated cardiovascular risk in patients with pa .
the aim of this study was to compare the levels of adma among patients with pa , controls with essential hypertension ( eh ) and healthy participants .
the study population consisted of 18 patients with pa , 18 controls with eh , and 18 healthy participants .
all studies were performed after obtaining a written informed consent form according to a protocol approved by the ethics committee of sofia medical university .
hypertensive patients were referred to the clinical center of endocrinology in sofia for diagnosis and treatment during june 2009 - june 2011 . in patients with eh other secondary forms of hypertension were ruled out , using medical history , physical examination , biochemical and hormonal investigations and imaging studies .
the diagnosis of pa was established , using the following algorithm : medications that can interfere with aldosterone and renin measurements ( beta - blockers , diuretics , angiotensin ii receptor blockers , angiotensin - converting enzyme inhibitors ) were discontinued for at least 7 - 10 days , and spironolactone was stopped for at least 45 days prior to blood sampling . centrally acting medicaments (
moxonodine , rilmenidine ) , alpha - blockers , and calcium channel blockers were used to treat hypertension .
blood samples for plasma renin activity ( pra ) and aldosterone were taken in the morning between 8 - 10 hours a.m. after the patient had been in a sitting position for 30 minutes .
the arr was then calculated and a confirmatory test , captopril test , was performed in patients with high arr ( above 750 pmol / l per ng / ml / h ) ( 10 ) and aldosterone above 416 pmol / l .
the diagnosis of pa was considered if the arr was > 970 after the oral administration of 50 mg of captopril , while the patient has been sitting for 90 minutes ( 14 ) .
plasma renin activity ( ng / ml / h ) was determined by quantitative determination of angiotensin i , using a commercially available radioimmunoassay kit ( diasorin s.p.a .
serum aldosterone was measured by radioimmunoassay ( immunotech , beckman coulter company , marseille , france ) and expressed in picomols per liter ( pmol / l ) .
the analytical sensitivity of the method was 0.20 ng / ml , inter - assay cv 7.5% , respectively , and intra - assay cv 5.4% . the cross reactivity with heptapeptide , angiotensin ii , and hexapeptide was below 0.02% .
we elaborated our own reference range for pra in a sitting position ( 0.3 - 3.5 ng / ml / h ) ( 15 ) . in cases in which pra was < 0.3 ng / ml / h or undetectable ,
serum aldosterone was measured by ria ( immunotech , beckman coulter ) . for intra - assay assessment ,
the analytical and functional sensitivity were 1.44 pg / ml , and 6.98 pg / ml , respectively . according to the manufacturer antibody used in this immunoassay was highly specific for aldosterone .
extremely low cross reactivities were found against other naturally occurring steroids ( cortisone , corticosterone , dheas , etc ) serum adma levels were determined , using competitive enzyme - linked immunosorbent assay ( elisa ) ( human adma elisa kit , promokine , heidelberg , germany ; thermo electron corporation : appliscan ) .
intra - assay and inter - assay cv for adma were 4.1 % and 4.5 % , respectively .
glucose was measured , using enzymatic reference method with hexokinase ( cobas integra , roche diagnostics ) .
intra - assay and inter - assay cv for glucose were 1.7% and 2.6% at 5.3
serum lipid parameters were assessed , using an enzymatic colorimetric method ( cobas integra , roche diagnostics ) . intra - assay and inter - assay cv for total cholesterol were 1.6% and 1.6% , respectively .
intra - assay and inter - assay cv for triglycerides were 1.6% and 3.7% , respectively .
intra - assay and inter - assay cv for hdl- cholesterol were 2.5 and 2.7% , respectively .
the inter - assay ( qc low and qc high ) was calculated , using mean analytic value of all plates according to the formula : cv ( % ) = standard deviation / mean value of analytic * 100 , which corresponds to ifcc protocol . according to generally accepted quality criteria cited above , cv% for inter- and intra - assay was acceptable , which ensured the reliability of the results .
statistical analysis was performed using spss 17.0 for windows ( chicago , illinois , usa ) .
gaussian - distributed data are shown as mean sd . student s t - test for independent samples was used to compare the equality of means for the two groups , and anova was used when testing three groups .
the study population consisted of 18 patients with pa , 18 controls with eh , and 18 healthy participants .
all studies were performed after obtaining a written informed consent form according to a protocol approved by the ethics committee of sofia medical university .
hypertensive patients were referred to the clinical center of endocrinology in sofia for diagnosis and treatment during june 2009 - june 2011 . in patients with eh other secondary forms of hypertension were ruled out , using medical history , physical examination , biochemical and hormonal investigations and imaging studies .
the diagnosis of pa was established , using the following algorithm : medications that can interfere with aldosterone and renin measurements ( beta - blockers , diuretics , angiotensin ii receptor blockers , angiotensin - converting enzyme inhibitors ) were discontinued for at least 7 - 10 days , and spironolactone was stopped for at least 45 days prior to blood sampling . centrally acting medicaments (
moxonodine , rilmenidine ) , alpha - blockers , and calcium channel blockers were used to treat hypertension .
blood samples for plasma renin activity ( pra ) and aldosterone were taken in the morning between 8 - 10 hours a.m. after the patient had been in a sitting position for 30 minutes .
the arr was then calculated and a confirmatory test , captopril test , was performed in patients with high arr ( above 750 pmol / l per ng / ml / h ) ( 10 ) and aldosterone above 416 pmol / l .
the diagnosis of pa was considered if the arr was > 970 after the oral administration of 50 mg of captopril , while the patient has been sitting for 90 minutes ( 14 ) .
plasma renin activity ( ng / ml / h ) was determined by quantitative determination of angiotensin i , using a commercially available radioimmunoassay kit ( diasorin s.p.a . , saluggia ( vc ) , italy ) .
serum aldosterone was measured by radioimmunoassay ( immunotech , beckman coulter company , marseille , france ) and expressed in picomols per liter ( pmol / l ) .
the analytical sensitivity of the method was 0.20 ng / ml , inter - assay cv 7.5% , respectively , and intra - assay cv 5.4% . the cross reactivity with heptapeptide , angiotensin ii , and hexapeptide was below 0.02% .
we elaborated our own reference range for pra in a sitting position ( 0.3 - 3.5 ng / ml / h ) ( 15 ) . in cases in which pra was < 0.3 ng / ml / h or undetectable ,
serum aldosterone was measured by ria ( immunotech , beckman coulter ) . for intra - assay assessment ,
for inter - assay assessment , the samples were assayed in duplicate in 10 different series .
the analytical and functional sensitivity were 1.44 pg / ml , and 6.98 pg / ml , respectively . according to the manufacturer antibody used in this immunoassay was highly specific for aldosterone .
extremely low cross reactivities were found against other naturally occurring steroids ( cortisone , corticosterone , dheas , etc ) serum adma levels were determined , using competitive enzyme - linked immunosorbent assay ( elisa ) ( human adma elisa kit , promokine , heidelberg , germany ; thermo electron corporation : appliscan ) .
intra - assay and inter - assay cv for adma were 4.1 % and 4.5 % , respectively .
glucose was measured , using enzymatic reference method with hexokinase ( cobas integra , roche diagnostics ) .
intra - assay and inter - assay cv for glucose were 1.7% and 2.6% at 5.3
serum lipid parameters were assessed , using an enzymatic colorimetric method ( cobas integra , roche diagnostics ) . intra - assay and inter - assay cv for total cholesterol were 1.6% and 1.6% , respectively . intra - assay and inter - assay cv for triglycerides were 1.6% and 3.7% , respectively .
intra - assay and inter - assay cv for hdl- cholesterol were 2.5 and 2.7% , respectively .
the inter - assay ( qc low and qc high ) was calculated , using mean analytic value of all plates according to the formula : cv ( % ) = standard deviation / mean value of analytic * 100 , which corresponds to ifcc protocol . according to generally accepted quality criteria cited above , cv% for inter- and intra - assay was acceptable , which ensured the reliability of the results .
statistical analysis was performed using spss 17.0 for windows ( chicago , illinois , usa ) .
student s t - test for independent samples was used to compare the equality of means for the two groups , and anova was used when testing three groups .
the baseline demographic and metabolic characteristics of those patients with primary aldosteronism , essential hypertension and normotensive controls are demonstrated in table 1 .
no significant differences were observed among the three groups in age , gender and fasting blood glucose .
patients with pa had higher body mass index and triglycerides and lower high- density ( hdl ) lipoprotein cholesterol compared to normotensive controls ( p = 0.001 , 0.016 , 0.002 , respectively ) .
patients with eh had higher levels of total cholesterol compared to patients with pa ( p = 0.024 ) .
patients with eh presented with significantly higher total cholesterol , low- density lipoprotein ( ldl ) cholesterol and triglycerides and lower hdl cholesterol than the control group ( p = 0.025 , p = 0.012 , p = 0.012 and p = 0.011 , respectively ) .
abbreviations : bmi , body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein .
adma levels were compared in patients with pa , eh and healthy controls ( figure 1 ) .
concentrations of adma were significantly higher in patients with pa than in healthy controls ( 0.49 0.09 vs. 0.43 0.05
there was no difference in adma levels between cases with pa and eh ( 0.49 0.09 vs. 0.48 0.08
the difference between participants with eh and the normotensive control group did not reach statistical significance ( p = 0.06 ) .
no significant correlations were found between adma and some metabolic parameters in patients with pa ( table 2 ) .
abbreviations : adma , asymmetric dimethylarginine ; bmi , body mass index ; hdl , high- density lipoprotein ; ldl , low - density lipoprotein .
endothelial dysfunction ( ed ) is characterized with a tendency to proinflammatory and prothrombotic state and reduced vasodilatation .
it is now well established that ed is related to the development of atherosclerosis ( 16 ) , myocardial infarction ( 17 ) , congestive heart failure ( 18 ) , diabetes mellitus ( 19 ) , and peripheral arterial diseases ( 20 ) .
one of the possible mechanisms leading to ed is decreased nitric oxide ( no ) bioavailability , which can be caused by decreased no synthesis . with regards to this mechanism , adma , which is an endogenous analogue of l - arginine , was firstly recognized in 1992 as a naturally occurring inhibitor of no synthase ( 21 ) .
moreover , elevated plasma adma concentration has been identified as a predictor of acute coronary events , and an independent risk factor for all - cause and cardiovascular mortality ( 8 , 9 ) .
although elevated concentrations of adma have been found in several cardiovascular and metabolic diseases ( 10 - 13 , 22 - 24 ) , to our knowledge , to date , there have been no reports on adma levels in cases with pa .
recent studies have demonstrated a higher frequency of cardiovascular events in pa compared to patients with eh ( 3 , 4 ) .
recent studies have demonstrated that aldosterone impairs endothelial function by suppression of the synthesis of no ( 25 - 27 ) .
experimental studies reported that the cytokine stimulated no synthesis in vascular smooth muscle cells is inhibited by aldosterone in a dose dependent manner ( 25 ) . recently , nishizaka et al .
( 26 ) reported a significant association between aldosterone and impaired endothelial function in human subjects , measured by flow - mediated arterial vasodilatation . in view of these findings
, we hypothesized that increased adma levels in pa could contribute to the impaired endothelial cell - dependent vasodilator responses .
our results confirmed the possible role of adma as a cardiovascular risk marker in pa since patients with pa had higher adma levels than healthy controls . on the other hand ,
numerous reports demonstrate that adma concentration is increased in patients with eh compared to healthy individuals ( 10 ) .
the latter findings raise the following question : is high adma concentration in pa related to the effect of aldosterone or to hypertension per se ? the comparable levels of adma in pa and eh suggest the responsible factor for adma dysregulation is hypertension itself rather than the specific aldosterone action .
indeed , several hypotheses for the elevation of adma in eh have been formulated , although the mechanism remains unclear .
( 28 ) have shown that adma release is enhanced by shear stress via activation of the nf - kappab pathway .
in addition , it is possible that the oxidative stress , a well- known feature of eh , induces elevated adma by suppressing the activity of its metabolizing enzyme - dimethylaminohydrolase ( 29 ) .
another possibility for the elevated adma concentration in pa and eh in our study was the increased ldl cholesterol and triglyceride levels , since it has been shown that ldl cholesterol decreases the activity of dimethylaminohydrolase .
on the other hand , we did not find any statistically significant correlation between adma and metabolic lipid parameters , which confirms the possibility , that hypertension , itself , rather than the deteriorated lipid profile , results in higher adma levels ( table 2 ) .
similar results were found in another study that we published recently , comparing adma in patients with pheochromocytoma and the control group of the present study ( 30 ) .
endothelial function was impaired in pheochromocytoma patients , as shown by the elevated circulating levels of adma .
the lack of association of these markers with glucose , cateholamines and lipid abnormalities as well as their comparable levels in patients with eh suggests that endothelial dysfunction may be related to hypertension itself .
this was the first report to investigate the adma levels in pa and provide insight into the possible mechanisms of cardiovascular damage in pa , which is the main strength of this study .
small sample size was the main study limitation , which was due to the restricted number of patients with pa .
adma is an endogenous analogue of l - arginine and a naturally occurring inhibitor of no synthase .
this was the first study to show elevated adma levels in patients with pa compared to normotensive controls .
our data raise the possibility for a role of adma dysregulation in the cardiovascular damage in pa . on the other hand ,
adma levels were similar in patients with pa and eh , which suggests that endothelial dysfunction is more likely related to hypertension per se , than to the specific etiology of elevated blood pressure .
this was the first report to investigate the adma levels in pa and provide insight into the possible mechanisms of cardiovascular damage in pa , which is the main strength of this study .
small sample size was the main study limitation , which was due to the restricted number of patients with pa .
adma is an endogenous analogue of l - arginine and a naturally occurring inhibitor of no synthase .
this was the first study to show elevated adma levels in patients with pa compared to normotensive controls .
our data raise the possibility for a role of adma dysregulation in the cardiovascular damage in pa . on the other hand ,
adma levels were similar in patients with pa and eh , which suggests that endothelial dysfunction is more likely related to hypertension per se , than to the specific etiology of elevated blood pressure . | backgroundrecent studies have revealed a higher rate of cardiovascular complications in primary aldosteronism ( pa ) compared to patients with essential hypertension ( eh ) .
asymmetric dimethylarginine ( adma ) is a marker of endothelial dysfunction that could contribute to increased cardiovascular risk in patients with pa.objectivesthe aim of this study was to compare the levels of adma among patients with pa , controls with eh and healthy participants .
methods : serum adma levels were determined , using commercially available competitive enzyme - linked immunosorbent assay.methodsserum adma levels were determined , using commercially available competitive enzyme - linked immunosorbent assay.resultspatients with pa had significantly higher concentrations of adma than healthy controls ( 0.488 0.085 vs. 0.433
0.053 mol / l , p = 0.027 ) .
no difference was found in adma levels between cases with pa and eh ( 0.488 0.085 vs. 0.476 0.075
mol / l , = 0.636 ) .
the difference between patients with eh and normotensive controls did not reach statistical significance ( p = 0.06).conclusionsthe lack of difference between adma levels in patients with pa and eh suggests that endothelial dysfunction is more likely related to hypertension per se than to the specific etiology of elevated blood pressure . |
the term primitive neuroectodermal tumors ( pnets ) were coined by hart and earle in 1973 .
these are a group of malignant neoplasms derived from the primitive neural crest and are highly malignant and mainly exist in the central nervous system ( cns ) , chest wall , lower extremities , trunk , kidney , orbit , and rarely in the spine .
in contrast , primary spinal cord tumors are very rare among children with an overall incidence rate of 0.26 per 100 person - years .
population - based data indicate that the most common histological types were meningiomas ( 29% ) , nerve sheath tumors ( 24% ) , and ependymomas ( 23% ) in this age group .
although multimodality treatments are available , the standard treatment for pnet is complete surgical excision followed by craniospinal radiotherapy . in this article
, we present a patient with primary intraspinal extradural pnet and describe its treatment regimen and outcome .
an 8-year - old female child had been experiencing back pain and weakness of both lower limbs for 1 month .
fifteen days before admission , she also noticed difficulty in standing . within days , the weakness progressed , and the patient was bedridden .
magnetic resonance imaging ( mri ) of the dorsal spine revealed a well - defined oval dorsal epidural lesion of size 6 cm 1 cm at d7d10 level with anterior compression of the cord .
it was hypointense on t1-weighted image ( t1-wi ) and t2-wi and no obvious contrast enhancement [ figure 1 ] .
magnetic resonance imaging study of the dorsal spine and paravertebral region showed well - defined oval epidural lesion of size 6 cm 1 cm from d7 to d10 levels along posterior aspect of cord with compression over the cord .
it was hypointense on t1/t2 with no obvious contrast enhancement in view of her symptoms , the patient was taken up for surgery , and d6d11 laminectomy was done .
a grayish - white , friable , soft , nonvascular tumor was seen extradurally which was adherent to the surrounding arachnoid and pia mater of cord .
histopathology revealed tumor composed of small round cells having scant cytoplasm forming diffuse sheets and islands separated by thin fibrous bands .
few pseudorosettes and occasional rosette formation seen along with areas of necrosis and vascular proliferation [ figure 3 ] .
on immunohistochemical ( ihc ) staining , we found diffuse , finely granular cytoplasmic material staining positive for synaptophysin throughout the tumor .
the final pathologic diagnosis of pnet was made along with some neuronal differentiation . in the absence of a primary brain tumor
( d ) tumor tissue removed en bloc is seen ( a ) tissue sections show densely packed sheets of primitive appearing cells ( h and e , 10 ) .
( b ) histopathological examination shows a highly cellular tumor , consisting mainly of small round - to - oval cells with hyperchromatic nuclei and remarkably scanty cytoplasm along with the presence of homer - wright pseudorosettes suggesting a round cell tumor .
note : highly cellular and poorly differentiated cells with mitosis figures and areas of necrosis with karyorrhexis ( h and e , 40 ) the patient was planned for postoperative radiotherapy and was referred to radiation oncology for adjuvant radiotherapy .
a treatment plan offering best therapeutic benefit both in terms of optimum dose to target tissue and maximum sparing of the uninvolved organs was selected .
the patient was given a total of 30 gy to the brain and spinal cord along with 8 g boost to involved area on dmx varian linear accelerator .
the patient tolerated the adjuvant radiotherapy well , and there was a marked improvement of pain and weakness symptoms 1 month after the radiotherapy .
in 1973 , hart and earle were the first to propose the term primitive neuroectodermal tumor for describing a neoplasm which was composed of 90%95% of undifferentiated cells which did not fulfill the diagnostic criteria for other entities .
rorke and becker and hinton in 1983 proved that all cns tumors composed of undifferentiated neuroepithelial cells are to be called pnets .
pnets are more common in children as in our case and can also occur in adults .
the mean age is between 5 and 77 years , and 80% of tumors occur in < 15 years of age .
there is a male predominance , being 1.44.8 times more common in males than in females .
pnet appears histologically as predominantly undifferentiated small , blue , round cell tumor with hyperchromatic nuclei , scanty cytoplasm , and frequent mitotic figures as was found in our case .
on ihc , variable positivity may be noted depending on differentiation ( neuronal , glial , or myogenic ) . in our case , it was positive for synaptophysin .
the tumor usually presents with nonspecific symptoms such as paraparesis , paresthesias , gait disturbance , and low back pain as in our case .
mri features of pnet are also usually nonspecific , with most of them being hyperintense on t2-wi and iso- to hypointense on t1-wi , with heterogeneous enhancement on postcontrast sequences . in our case , it was hypointense on t1-wi and t2-wi and had no obvious contrast enhancement .
suggest that although imaging findings are not specific , the diagnosis could be suggested when mri shows extradural large , well - circumscribed mass extending out from the intervertebral foramen and invading paraspinal soft tissues or vertebral bones in a young patient .
after adjuvant chemotherapy , survival rates may be as high as 82% at 5 years .
the use of chemotherapy for treating patients with pnets outside the cerebellum , particularly spinal pnets , has been rather inconsistent apart from the standard use of surgical resection and radiotherapy .
it is probably because such tumors are rare , and there is a lack of prospective protocol to address the benefits of specific chemotherapeutic regimens .
survival in spinal pnet ranges from 3 months to more than 3 years from the time of diagnosis .
pnet should be included in differential diagnosis of spinal tumors , especially in children and young adults having an intraspinal mass . as there is no standard protocol for the management of spinal pnets , we advise complete resection with postoperative chemoradiation .
primary intraspinal pnet subtyping after histopathological and ihc studies helps us plan an optimal treatment strategy for these aggressive tumors .
| primitive neuroectodermal tumors ( pnets ) are aggressive childhood malignancies and are difficult to treat .
primary intraspinal pnets are rare .
these patients have poor prognosis with short survival time even after surgery and chemoradiation . as there are
no standard guidelines exist for the management of these tumors , a multidisciplinary approach has been employed with varying success . according to the review of literature ,
only few cases of primary intraspinal extradural pnets have been reported .
herein , author has described a case of intraspinal , extradural pnet . |
a total of 38 patients with chronic painful dpn ( neuropathy total symptom score 6 > 4 and < 16 ) for at least 6 months with stable glycemic control ( a1c < 11% ) were assessed .
those with persistent pain , despite an adequate trial of tricyclic antidepressants , were recruited .
three modalities of pain ( superficial , deep , and muscular pain ) were assessed daily using a 100-mm visual analog scale ( vas ) .
the dose of study medication was titrated over 2 weeks , followed by a 10-week maintenance phase . at baseline
, depression was assessed using the seven - item depression subscale of the hospital anxiety and depression scale ( hads - d ) ( 3 ) .
improvement in pain , as assessed by the pain diary and neuropathic pain scale ( nps ) questionnaire , was used as the primary outcome measure .
study end point was the final week mean pain and nps score while taking the maximum tolerated dose of study medication .
a total pain score ( tps ) ( average score of all three pain modalities ) was also calculated .
secondary outcome measure was quality of life ( qol ) , assessed by mcgill pain and qol ( 5 ) , sf-36 health survey ( 6 ) , and euroqol ( 7 ) questionnaires .
sativex ( tetrahydrocannabinol [ 27 mg / ml ] and cannabidiol [ 25 mg / ml ] ) and its matching placebo were presented as a pump - action spray .
doses were administered sublingually in divided doses up to four times a day . an intent - to - treat analysis was undertaken .
differences in subgroup baseline characteristics were correlated to the outcome and adjustments performed at a coefficient > 0.50 .
multiple linear regression was used for a normal distribution , while skewed distribution was initially transformed .
hoc analysis , patients were divided into individuals with depression ( hads - d score 10 ) and no depression ( hads - d score < 10 ) .
using ancova , we compared mean change in tps from baseline between these groups . the interaction between depression and treatment
each treatment arm was divided into patients with and without depression , and outcomes were compared using an independent sample t test .
differences in subgroup baseline characteristics were correlated to the outcome and adjustments performed at a coefficient > 0.50 .
multiple linear regression was used for a normal distribution , while skewed distribution was initially transformed .
hoc analysis , patients were divided into individuals with depression ( hads - d score 10 ) and no depression ( hads - d score < 10 ) . using ancova ,
each treatment arm was divided into patients with and without depression , and outcomes were compared using an independent sample t test .
we excluded one placebo - treated patient from the intent - to - treat analysis ( n = 29 ) because of a protocol violation .
covariates used in the analysis were duration of diabetes , baseline scores , age , and sex .
there was no significant difference in mean change tps between sativex and placebo ( p = 0.40 ; sem 9.5 ; 95% ci 11.3 to 27.8 ) at end point .
similarly , there was no difference in mean change in superficial ( p = 0.72 ; 9.1 ; 15.3 to 21.93 ) , deep ( p = 0.38 ; 10.5 ; 12.2 to 30.8 ) , and muscular ( p = 0.26 ; 10.3 ; 9.15 to 33.0 ) pain vas .
differences in nps did not reach statistical significance ( p = 0.62 ; 7.8 ; 20.1 to 12.1 ) .
eight ( 53% ) sativex - treated patients responded ( defined as 30% total pain vas improvement ) versus nine ( 64% ) placebo patients ( p = 0.55 , odds ratio 0.63 , 95% ci 0.142.82 ) ( table 1 ) . demographics and primary and secondary outcome measures data are n or means sd unless otherwise indicated .
the mcgill pain questionnaire showed no difference in sensory scale ( p = 0.65 ; sem 3.3 ; 95% ci 5.39 to 8.44 ) , affective scale ( p = 0.81 ; 1.3 ; 3.0 to 2.4 ) , vas ( p = 0.24 ; 1.0 ; 0.91 to 3.4 ) , and present pain intensity ( p = 0.57 ; 0.53 ; 0.79 to 1.4 ) between study cohorts .
euroqol and sf-36 questionnaires showed improvement in both groups , but differences between groups were not statistically significant ( table 1 ) .
mean hads - d for patients with depression ( n = 10 ) and no depression ( n = 18 ) were 13.4 3.5 ( means sd ) and 5.94 2.2 , respectively . patients with depression had significantly higher baseline tps ( 62.3 22.1 vs. 43.4 24.3 ; p = 0.05 ) and greater tps improvement ( 31.6 24.2 vs. 10.7 25.0 ; p = 0.04 , sem 9.8 , 95% ci 0.5441.1 ) compared with those without depression .
however , there was a significant main effect of depression on tps ( p = 0.05 ) , suggesting that in both treatment arms , patients who were depressed were more likely to respond to intervention : sativex arm , depressed ( 36.7 28.6 ) vs. nondepressed ( 4.9 14.4 ) , p = 0.02 , 56.5 to 7.2 ; placebo arm , 26.5 20.7 vs. 17.3 33.1 , p = 0.60 , 45.9 to 27.6 .
covariates used in the analysis were duration of diabetes , baseline scores , age , and sex .
there was no significant difference in mean change tps between sativex and placebo ( p = 0.40 ; sem 9.5 ; 95% ci 11.3 to 27.8 ) at end point .
similarly , there was no difference in mean change in superficial ( p = 0.72 ; 9.1 ; 15.3 to 21.93 ) , deep ( p = 0.38 ; 10.5 ; 12.2 to 30.8 ) , and muscular ( p = 0.26 ; 10.3 ; 9.15 to 33.0 ) pain vas .
differences in nps did not reach statistical significance ( p = 0.62 ; 7.8 ; 20.1 to 12.1 ) .
eight ( 53% ) sativex - treated patients responded ( defined as 30% total pain vas improvement ) versus nine ( 64% ) placebo patients ( p = 0.55 , odds ratio 0.63 , 95% ci 0.142.82 ) ( table 1 ) . demographics and primary and secondary outcome measures data are n or means sd unless otherwise indicated .
the mcgill pain questionnaire showed no difference in sensory scale ( p = 0.65 ; sem 3.3 ; 95% ci 5.39 to 8.44 ) , affective scale ( p = 0.81 ; 1.3 ; 3.0 to 2.4 ) , vas ( p = 0.24 ; 1.0 ; 0.91 to 3.4 ) , and present pain intensity ( p = 0.57 ; 0.53 ; 0.79 to 1.4 ) between study cohorts .
euroqol and sf-36 questionnaires showed improvement in both groups , but differences between groups were not statistically significant ( table 1 ) .
mean hads - d for patients with depression ( n = 10 ) and no depression ( n = 18 ) were 13.4 3.5 ( means sd ) and 5.94 2.2 , respectively . patients with depression had significantly higher baseline tps ( 62.3 22.1 vs. 43.4 24.3 ; p = 0.05 ) and greater tps improvement ( 31.6 24.2 vs. 10.7 25.0 ; p = 0.04 , sem 9.8 , 95% ci 0.5441.1 ) compared with those without depression .
however , there was a significant main effect of depression on tps ( p = 0.05 ) , suggesting that in both treatment arms , patients who were depressed were more likely to respond to intervention : sativex arm , depressed ( 36.7 28.6 ) vs. nondepressed ( 4.9 14.4 ) , p = 0.02 , 56.5 to 7.2 ; placebo arm , 26.5 20.7 vs. 17.3 33.1 , p = 0.60 , 45.9 to 27.6 .
despite being common , there are few effective treatments that provide symptomatic relief for painful dpn ( 8) . for centuries , cannabinoids have been consumed for their analgesic properties and more recently studied in other neuropathic conditions ( 9 ) . in this study , when compared with placebo , sativex failed to show statistically significant improvements in primary and secondary outcome measures .
patients with depression had higher baseline pain scores and were also more likely to respond favorably to intervention , regardless if sativex or placebo .
most painful dpn trials to date either have not screened for depression or exclude individuals who have it ( 10,11 ) .
as in a number of recent studies , there was a large placebo effect that may have led to a failure to show differences in outcome measures ( 12 ) .
this may provide an insight into the nature of pain in dpn and the placebo effect .
there is a need for more robust and objective end points for use in clinical trials of painful dpn .
also , it was felt ethically inappropriate to discontinue treatments from which patients may be benefiting .
the use of specific painful dpn qol questionnaires ( 13 ) may have captured subtle changes missed by the generic ones used in this study . finally ,
while the search for therapeutic agents to halt or reverse the neuropathic process continues , more effective treatments are required that provide better symptom control with fewer side effects .
the assessment of depression may be important when designing future clinical trials into painful dpn . | objectiveto assess the efficacy of sativex , a cannabis - based medicinal extract , as adjuvant treatment in painful diabetic peripheral neuropathy ( dpn).research design and methodsin this randomized controlled trial , 30 subjects with painful dpn received daily sativex or placebo .
the primary outcome measure was change in mean daily pain scores , and secondary outcome measures included quality - of - life assessments.resultsthere was significant improvement in pain scores in both groups , but mean change between groups was not significant .
there were no significant differences in secondary outcome measures .
patients with depression had significantly greater baseline pain scores that improved regardless of intervention.conclusionsthis first - ever trial assessing the efficacy of cannabis has shown it to be no more efficacious than placebo in painful dpn .
depression was a major confounder and may have important implications for future trials on painful dpn . |
complications during central venous catheter placement are uncommon and their incidence has decreased further with the use of ultrasound ( usg ) guidance for venous puncture .
common vascular complications described during this procedure include common carotid artery ( cca ) injury leading to bleeding , dissection , pseudoaneurysm , and thrombosis , internal jugular vein ( ijv ) dissection , and venous pseudoaneurysm formation .
a 50-year - old lady with advanced chronic renal disease was referred to the interventional radiology ( ir ) suite for placement of an ijv dialysis access catheter .
her international normalized ratio ( inr ) was 1.5 and serum platelet count was 80,000/mm . before referral , unsuccessful attempts to cannulate the right ijv access were made in the dialysis laboratory . in the ir suite ,
a screening usg of the neck ( logiq e ; ge healthcare , milwaukee , wisconsin , usa ; 10 mhz linear probe ) revealed a moderate hematoma in the lower right neck compressing the ijv , with complete thrombosis of the right ijv .
the left ijv was also partially thrombosed with thrombus distending the vein , suggesting an acute thrombus . as there were relatively large and well - formed collaterals in the right lower neck draining into the subclavian vein , we did an usg - guided puncture and venogram of one of these collaterals , but decided not to attempt insertion of a dialysis cannula due to the tortuous anatomy .
as the patient had no other significant venous access for dialysis ( femoral veins were also thrombosed and partly recanalized ) , we decided to recanalize the left ijv and insert a dialysis catheter .
a non - thrombosed segment of left ijv was punctured under usg guidance , the thrombosed segment traversed with a 0.035-inch , 180-cm j - tip glide wire ( radifocus ; terumo , tokyo , japan ) , and balloon angioplasty was done with a 10 mm x 2 cm balloon ( opta pro pta dilatation catheter , cordis , miami , usa ) following which antegrade flow was established in the left ijv .
we placed an 11-fr ijv dialysis catheter ( bard , salt lake city , utah , usa ) with its tip within the left subclavian vein .
one day after insertion of the left ijv dialysis catheter , the patient was found to have pain and an increasing swelling on the right side of the neck with ecchymosis .
doppler evaluation of the swelling showed a large hematoma anterior to the neck vessels with a pseudoaneurysm formation [ figure 1a ] .
the spectral waveforms of the right cca , right internal carotid artery ( ica ) , and the right ijv were normal .
( a ) doppler image showing aliasing and low - resistance arterial flow within the pseudoaneurysm ( arrow ) ( b ) axial contrast - enhanced ct of the neck showing a hematoma ( asterisk ) with contrast pooling anterior to the right ijv ( long arrow ) and the right cca ( small arrow ) ( c ) coronal ct image of the neck showing contrast pooling ( arrow ) in the right lower neck computed tomographic ( ct ) angiography of the neck vessels ( philips brilliance 6 ct scanner , philips , cleveland , ohio , usa ) showed a large hematoma in the right lower neck with an area of contrast extravasation adjacent to the hematoma , corresponding to the pseudoaneurysm .
the pseudoaneurysm was in close relation to the right ijv and right cca , but no definitive communication with either vessel could be demonstrated on the ct angiogram [ figure 1b and c ] . as the pseudoaneurysm was recently formed , with a narrow neck on doppler , we attempted usg - guided graded compression of the pseudoaneurysm ( 15 min of compression with slow release of pressure at the end of this period to assess outcome , with a 1 min break after which the compression was repeated once more ) for two consecutive days in an attempt to obliterate the pseudoaneurysm .
but the pseudoaneurysm persisted . as the hematoma and ecchymosis in the right lower neck continued to enlarge in size clinically
, we decided to perform a digital subtraction angiogram ( dsa ) evaluation of the neck vessels . under local anesthesia , through a right transfemoral arterial access ( 4 fr , 11 cm introducer sheath ; cordis , miami , fl , usa ) , a right cca angiogram was done ( artis zee biplane ; siemens , erlangen , germany ) using a 100-cm , 4-fr head - hunter catheter ( radifocus ; terumo , tokyo , japan ) .
common and selective right external carotid artery angiograms showed a small pseudoaneurysm arising from the sternocleidomastoid branch of the right superior thyroid artery , with associated arteriovenous fistula [ figure 2a and b ] draining into a venous collateral .
the involved artery was superselectively cannulated with a microcatheter ( progreat microcatheter system ; terumo , tokyo , japan ) and embolized with two 2 mm 2 cm , 0.18-inch fibered platinum pushable coils ( hilal ; cook , washington , usa ) .
post - embolization check angiogram showed complete occlusion of the pseudoaneurysm and the arteriovenous fistula [ figure 2c ] .
the neck hematoma started reducing over the next 48 h and was almost completely resolved clinically by the end of 1 week .
( a ) right eca angiogram , lateral projection ( late arterial phase ) showing a pseudoaneurysm ( long arrow ) arising from a branch of the right superior thyroid artery .
early filling of a venous channel is also seen ( short arrow ) , confirming the presence of a fistula ( b ) superselective angiogram of the right superior thyroid artery demonstrating the pseudoaneurysm and the arteriovenous fistula ( c ) post - coiling angiogram of the right superior thyroid artery showing complete occlusion of the pseudoaneurysm and the arteriovenous fistula
the right ijv access is the standard practice for placing a central venous catheter / ijv catheter due to an in - line flow - directed placement and less chances of pneumothorax compared to a subclavian access .
inadvertent arterial puncture during ijv cannulation is rare ( 0.1 - 0.4% ) and usually involves injury to the cca .
the most common etiology leading to carotid artery injury and pseudoaneurysm formation is neck trauma , with majority of the cases involving the ica .
very few cases of external carotid artery pseudoaneurysm have been described , either due to trauma or due to iatrogenic injury .
arterial pseudoaneurysms usually increase in size over a course of few days and present as a pulsatile mass . however , our case manifested as a neck swelling and ecchymosis ( due to subcutaneous bleeding ) , which may have been due to the relatively small pseudoaneurysm .
duplex usg ( doppler ) is a useful screening modality for initial assessment of an arterial injury .
, doppler and ct demonstrated the pseudoaneurysm , but precise localization of the injured vessel could be ascertained only on conventional angiography .
treatment options for pseudoaneurysm closure include surgical ligation , usg - guided compression , percutaneous thrombin injection , and endovascular techniques such as coil embolization and injection of liquid embolic agents .
usg - guided compression did not succeed in our case , probably due to the lack of an underlying hard bony structure against which the pseudoaneurysm neck could have been compressed .
we attempted it first only because it was a relatively non - invasive treatment option .
however , usg - guided compression is not the preferred choice of treatment for neck pseudoaneurysms .
usg - guided thrombin injection for pseudoaneurysm closure which was first described in 1986 is potentially risky in the head and neck territory due to the possibility of inadvertent thrombin embolization to the intracranial circulation . in our case ,
as the vascular anatomy was favorable , the injured artery could be selectively cannulated and embolized with coils .
to our knowledge , there are no other case reports of superior thyroid artery branch pseudoaneurysm and arteriovenous fistula following attempted ijv access .
this case report also highlights the importance of recognizing a vascular complication after jugular access . | vascular injury during common jugular venous ( ijv ) access is a rare complication , usually involving injury to the common carotid artery .
we describe a previously unreported complication of iatrogenic injury of ijv access involving a branch of the superior thyroid artery , and its endovascular management . |
before the start of sample preparation one needs to program the automatic microwave tissue processor for electron microscopy ( leica em amw ; leica microsystems , vienna , austria ) with the following protocols ( table 1 ) for microwave assisted sample preparation for tem :
table 1 .
the different columns show ( from left to right ) : vial number ( vial nr . )
represents the order in which the vial is loaded into the carousel of the processor.step of the actual process.reagents in the vials.duration of the actual step.maximum temperature which is reached in the vial before the microwave irradiation is turned off.microwave irradiation setting : continuous= rapid temperature increase , holding the set temperature ; slope = gentle temperature increase , final temperature reached at the end ; pulsed = rapid temperature increase , power turned off until the temperature dropped 5c , power resumed to reach temperature.maximum power of the microwave irradiation .
) represents the order in which the vial is loaded into the carousel of the processor .
maximum temperature which is reached in the vial before the microwave irradiation is turned off .
microwave irradiation setting : continuous= rapid temperature increase , holding the set temperature ; slope = gentle temperature increase , final temperature reached at the end ; pulsed = rapid temperature increase , power turned off until the temperature dropped 5c , power resumed to reach temperature .
freshly prepare the solutions for the different steps described in the sample preparation protocol ( for agar 100 epoxy resin see 1.7 ) , fill them into the designated vials according to the programmed protocol ( table 1 ) , load the vials on the carousel , then insert the carousel into the microwave tissue processor , and finally load the first vial into the mono - mode chamber . cut out small sections of leaves ( 1 mm ) from nicotiana tabacum infected with tobacco mosaic virus ( tmv ) with a razor blade on a modeling wax plate in a drop of 3% glutaraldehyde ( agar scientific ltd . , stansted , england ) in 60 mm srensen phosphate buffer ( ph 7.2 ) at room temperature .
transfer the sections with fine tweezers immediately into the designated baskets with a mesh width of approximately 200m .
stack the baskets on top of each other and insert them into the mono - mode chamber .
care must be taken that the samples are constantly covered with fixative solution during loading and stacking of the baskets so that they do not dry out .
start the previously programmed microwave assisted sample preparation protocol for fixation , dehydration and infiltration . while sample preparation is performed automatically by the microwave tissue processor
continue with negative staining with the remaining plant material as described in section 3 ( negative staining ) .
freshly prepare agar 100 epoxy resin by mixing the following components as described : fill 24 g agar 100 , 16 g dodecenyl succinic anhydride , and 10 g methyl nadic anhydride ( for all components see agar scientific ltd .
, stansted , england ) in a plastic cup , heat it to 40c and mix it well .
fill agar 100 epoxy resin into the designated polymerization forms just before the sample preparation protocol comes to an end ( e.g. during step 22 in table 1 ) . after the protocol is finished ( after step 22 in table 1 )
release the stacked baskets containing the infiltrated samples from the mono - mode chamber into the last vial of the carousel . remove the carousel from the microwave device , unstack the baskets and load them by using fine tweezers into the designated polymerization forms .
care must be taken that the samples are always covered with agar 100 epoxy resin during unstacking and loading so that they do not dry out .
care must be taken that the samples are always covered with agar 100 epoxy resin during stacking and loading so that they do not dry out .
remove previously used vials from the carousel of the microwave tissue processor , load it with the stacked baskets and insert the carousel into the microwave tissue processor . start the previously programmed polymerization protocol ( table 1 ) .
while polymerization is carried out automatically by the microwave tissue processor , examine negatively stained grids with a transmission electron microscope [ e.g. philips cm10 tem , fei ( formerly philips ) , eindhoven , the netherlands ] and perform image analysis as described in section 3 and 4 ( negative staining and image analysis ) .
after the protocol is finished remove the polymerization forms from the mono - mode chamber , unstack the polymerization forms and remove the polymerized blocks containing the samples .
insert one or more blocks into separate sample holders for ultrathin sectioning with the sample on top sticking about 1 cm out of the holder .
trim the block with a specimen trimmer for tem ( e.g. leica reichert ultratrim ; leica microsystems ) so that a block face of max .
1 mm in length and 200m in width which contains as much leaf material as possible is achieved ( block face size might need to be adjusted to the size of the diamond knife ) . section the block with an ultramicrotome ( e.g. reichert leica ultracut s ; leica microsystems ) by using a diamond knife at a knife angle of 45 ( e.g. diatome ultra 45 , grpl , tulln , austria ) .
section thickness should be adjusted to about 70 to 90 nm and the cutting speed should be around 1mm / s
post - stain the sections on the grid with lead citrate ( agar scientific ltd . ;
1.1 g lead citrate dissolved in 42ml double distilled water and 8ml 1n naoh ) for 5 minutes in a petri dish partly filled with naoh to create a co2-free environment and for 15 minutes with 1% uranyl acetate ( agar scientific ltd . ) dissolved in distilled water at room temperature .
wash the grids with distilled water for 1 minute in between each post - staining step .
examine sections with a transmission electron microscope ( e.g. philips cm10 tem , fei , eindhoven , the netherlands ) .
harvest about 100 mg of tmv - infected leaf material and prepare crude sap by homogenizing the material for 2 minutes with a razor blade on a microscope slide in 100l of 60 mm sfrensen phosphate buffer ( ph 7.2 ) .
transfer 20l of the resulting homogenate onto the first well of a teflon coated microscope slide with 4 or more wells ( alternatively it is also possible to transfer the homogenate on a piece of parafilm ) . place a formvar coated grid on top of the homogenate with the formvar ( agar scientific ltd . )
wash the grid 2 times for 2 minutes each by placing the grid on top of two drops of 200l 60 mm srensen phosphate buffer ( ph 7.2 ) .
incubate the grid for 1 minute with a freshly prepared 2% phosphotungstic acid ( agar scientific ltd . )
examine the grid with a transmission electron microscope ( e.g. philips cm10 tem ; fei , eindhoven , the netherlands ) .
take at least 10 images of randomly chosen negatively stained virions ( at least 10 or more virions should be visible on each image ) with the transmission electron microscope at a primary magnification of 21000x or higher .
measure the length and width of at least 100 randomly chosen single virus particles on the micrographs taken from the negatively stained samples by using any image analysis computer software [ e.g. cell d ( olympus , life and material science europe gmbh , hamburg , germany ) with the particle analysis tool or optimas 6.5.1- ( media cybernetics inc . ,
calculate means and standard deviations in order to achieve an average length and width of the virus particles visualized by negative staining methods and tem .
compare length and ultrastructural features ( obtained in 2.5 ) with sizes of viruses and ultrastructural alterations induced by virus diseases known in the literature in order to clearly identify the virus diseases .
after microwave assisted sample preparation typical tmv - induced ultrastructural alterations such as large areas containing virions aligned in parallel form could be observed with the transmission electron microscope in the cytosol of infected nicotiana tabacum cells ( fig 1a ) .
additionally , in the crude sap of tmv - infected leaves tmv particles could be observed as flexuous , rod - shaped structures after negative staining ( fig 1b ) .
image analysis of 100 virus particles revealed an average size for tmv of 280 nm in length and 17 nm in width ( fig 2 ) .
the ultrastructure in tmv - infected cells and the size of virions observed in this study were found to be in accordance with tmv - induced ultrastructural properties in tobacco and the size range of tmv particles previously reported in the literature .
a ) image shows the ultrastructure of tmv - infected mesophyll leaf cells of nicotiana tabacum after microwave assisted plant sample preparation .
c = chloroplast with starch ( st ) , m = mitochondrion , n = nucleus , v = vacuole , bar=2m .
b ) image shows virions which were detected by negative staining in the sap of infected leaves .
relative distribution of length and width of tmv - particles after negative staining the sap of infected leaves as they appeared in the electron microscope .
mean values ( mean length / width standard deviation ) were calculated from 100 virions .
before the start of sample preparation one needs to program the automatic microwave tissue processor for electron microscopy ( leica em amw ; leica microsystems , vienna , austria ) with the following protocols ( table 1 ) for microwave assisted sample preparation for tem :
table 1 .
the different columns show ( from left to right ) : vial number ( vial nr . )
represents the order in which the vial is loaded into the carousel of the processor.step of the actual process.reagents in the vials.duration of the actual step.maximum temperature which is reached in the vial before the microwave irradiation is turned off.microwave irradiation setting : continuous= rapid temperature increase , holding the set temperature ; slope = gentle temperature increase , final temperature reached at the end ; pulsed = rapid temperature increase , power turned off until the temperature dropped 5c , power resumed to reach temperature.maximum power of the microwave irradiation .
) represents the order in which the vial is loaded into the carousel of the processor .
maximum temperature which is reached in the vial before the microwave irradiation is turned off .
microwave irradiation setting : continuous= rapid temperature increase , holding the set temperature ; slope = gentle temperature increase , final temperature reached at the end ; pulsed = rapid temperature increase , power turned off until the temperature dropped 5c , power resumed to reach temperature .
freshly prepare the solutions for the different steps described in the sample preparation protocol ( for agar 100 epoxy resin see 1.7 ) , fill them into the designated vials according to the programmed protocol ( table 1 ) , load the vials on the carousel , then insert the carousel into the microwave tissue processor , and finally load the first vial into the mono - mode chamber . cut out small sections of leaves ( 1 mm ) from nicotiana tabacum infected with tobacco mosaic virus ( tmv ) with a razor blade on a modeling wax plate in a drop of 3% glutaraldehyde ( agar scientific ltd . , stansted , england ) in 60 mm srensen phosphate buffer ( ph 7.2 ) at room temperature .
transfer the sections with fine tweezers immediately into the designated baskets with a mesh width of approximately 200m .
stack the baskets on top of each other and insert them into the mono - mode chamber .
care must be taken that the samples are constantly covered with fixative solution during loading and stacking of the baskets so that they do not dry out .
start the previously programmed microwave assisted sample preparation protocol for fixation , dehydration and infiltration . while sample preparation is performed automatically by the microwave tissue processor
continue with negative staining with the remaining plant material as described in section 3 ( negative staining ) .
freshly prepare agar 100 epoxy resin by mixing the following components as described : fill 24 g agar 100 , 16 g dodecenyl succinic anhydride , and 10 g methyl nadic anhydride ( for all components see agar scientific ltd .
, stansted , england ) in a plastic cup , heat it to 40c and mix it well .
fill agar 100 epoxy resin into the designated polymerization forms just before the sample preparation protocol comes to an end ( e.g. during step 22 in table 1 ) . after the protocol is finished ( after step 22 in table 1 )
release the stacked baskets containing the infiltrated samples from the mono - mode chamber into the last vial of the carousel . remove the carousel from the microwave device , unstack the baskets and load them by using fine tweezers into the designated polymerization forms .
care must be taken that the samples are always covered with agar 100 epoxy resin during unstacking and loading so that they do not dry out .
care must be taken that the samples are always covered with agar 100 epoxy resin during stacking and loading so that they do not dry out .
remove previously used vials from the carousel of the microwave tissue processor , load it with the stacked baskets and insert the carousel into the microwave tissue processor . start the previously programmed polymerization protocol ( table 1 ) .
while polymerization is carried out automatically by the microwave tissue processor , examine negatively stained grids with a transmission electron microscope [ e.g. philips cm10 tem , fei ( formerly philips ) , eindhoven , the netherlands ] and perform image analysis as described in section 3 and 4 ( negative staining and image analysis ) .
after the protocol is finished remove the polymerization forms from the mono - mode chamber , unstack the polymerization forms and remove the polymerized blocks containing the samples .
insert one or more blocks into separate sample holders for ultrathin sectioning with the sample on top sticking about 1 cm out of the holder .
trim the block with a specimen trimmer for tem ( e.g. leica reichert ultratrim ; leica microsystems ) so that a block face of max
. 1 mm in length and 200m in width which contains as much leaf material as possible is achieved ( block face size might need to be adjusted to the size of the diamond knife ) . section the block with an ultramicrotome ( e.g. reichert leica ultracut s ; leica microsystems ) by using a diamond knife at a knife angle of 45 ( e.g. diatome ultra 45 , grpl , tulln , austria ) .
section thickness should be adjusted to about 70 to 90 nm and the cutting speed should be around 1mm / s .
post - stain the sections on the grid with lead citrate ( agar scientific ltd . ; 1.1 g lead citrate dissolved in 42ml double distilled water and 8ml 1n naoh ) for 5 minutes in a petri dish partly filled with naoh to create a co2-free environment and for 15 minutes with 1% uranyl acetate ( agar scientific ltd . )
wash the grids with distilled water for 1 minute in between each post - staining step .
examine sections with a transmission electron microscope ( e.g. philips cm10 tem , fei , eindhoven , the netherlands ) .
harvest about 100 mg of tmv - infected leaf material and prepare crude sap by homogenizing the material for 2 minutes with a razor blade on a microscope slide in 100l of 60 mm sfrensen phosphate buffer ( ph 7.2 ) .
transfer 20l of the resulting homogenate onto the first well of a teflon coated microscope slide with 4 or more wells ( alternatively it is also possible to transfer the homogenate on a piece of parafilm ) .
place a formvar coated grid on top of the homogenate with the formvar ( agar scientific ltd . )
wash the grid 2 times for 2 minutes each by placing the grid on top of two drops of 200l 60 mm srensen phosphate buffer ( ph 7.2 ) .
incubate the grid for 1 minute with a freshly prepared 2% phosphotungstic acid ( agar scientific ltd . )
solution in 60 mm srensen phosphate buffer ( ph 6.5 ) . remove the grid and allow it to air dry in a grid box . examine the grid with a transmission electron microscope ( e.g. philips cm10 tem ; fei , eindhoven , the netherlands ) . take at least 10 images of randomly chosen negatively stained virions ( at least 10 or more virions should be visible on each image ) with the transmission electron microscope at a primary magnification of 21000x or higher .
measure the length and width of at least 100 randomly chosen single virus particles on the micrographs taken from the negatively stained samples by using any image analysis computer software [ e.g. cell d ( olympus , life and material science europe gmbh , hamburg , germany ) with the particle analysis tool or optimas 6.5.1- ( media cybernetics inc . ,
bethesda , maryland , usa ) ] . calculate means and standard deviations in order to achieve an average length and width of the virus particles visualized by negative staining methods and tem .
compare length and ultrastructural features ( obtained in 2.5 ) with sizes of viruses and ultrastructural alterations induced by virus diseases known in the literature in order to clearly identify the virus diseases .
after microwave assisted sample preparation typical tmv - induced ultrastructural alterations such as large areas containing virions aligned in parallel form could be observed with the transmission electron microscope in the cytosol of infected nicotiana tabacum cells ( fig 1a ) .
additionally , in the crude sap of tmv - infected leaves tmv particles could be observed as flexuous , rod - shaped structures after negative staining ( fig 1b ) .
image analysis of 100 virus particles revealed an average size for tmv of 280 nm in length and 17 nm in width ( fig 2 ) .
the ultrastructure in tmv - infected cells and the size of virions observed in this study were found to be in accordance with tmv - induced ultrastructural properties in tobacco and the size range of tmv particles previously reported in the literature .
a ) image shows the ultrastructure of tmv - infected mesophyll leaf cells of nicotiana tabacum after microwave assisted plant sample preparation .
c = chloroplast with starch ( st ) , m = mitochondrion , n = nucleus , v = vacuole , bar=2m .
b ) image shows virions which were detected by negative staining in the sap of infected leaves .
relative distribution of length and width of tmv - particles after negative staining the sap of infected leaves as they appeared in the electron microscope .
mean values ( mean length / width standard deviation ) were calculated from 100 virions .
microwave assisted plant sample preparation for tem has been proven to supply fast and reliable ultrastructural data within a few hours .
the fine structural preservation of organelles and membranes achieved with the method used in this study was similar to conventional and cryofixed samples with the advantage of a massive reduction in sample fixation and embedding time from 3 days or longer to about 2 hours .
the method described in this study combined microwave assisted plant sample preparation for tem with negative staining methods which allowed a clear and rapid identification of tmv - induced ultrastructural alterations and the viral agent itself .
tmv induced ultrastructural alterations could be investigated after trimming , sectioning and post - staining within about 4 hours after the beginning of fixation in the transmission electron microscope . using
the fully automatically specimen preparation mode freed the researcher to conduct negative staining in the interim in order to determine the size and width of the viral agent .
thus , we can conclude that this method allows a clear and rapid diagnosis of plant virus diseases in about half a day which is of great importance for future use in agriculture and scientific experiments in plant phytopathology . as this method
could also be used for the rapid diagnosis of animal and human diseases it has a large potential for future application in medical and veterinary pathology .
| investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants .
with conventional sample preparation for transmission electron microscopy ( tem ) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases .
microwave fixation can be used to drastically reduce sample preparation time for tem investigations with similar ultrastructural results as observed after conventionally sample preparation3 - 5 .
many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for tem investigations5 - 8 . in this study
we demonstrate on tobacco mosaic virus ( tmv ) infected nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for tem .
we have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6 - 8 and are therefore more time and labor consuming .
as sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining tmv - infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding . |
in the development and patterning of embryonic and adult tissues , secreted signaling molecules of the wnt , fgf , hedgehog , and transforming growth factor ( tgf- ) families can act as morphogens to activate different transcriptional programs along a signaling gradient ( perrimon et al . , 2012 ) .
ideas of how morphogens impart spatial information have been dominated by the assumption that these molecules form concentration gradients by diffusion , inducing dose - dependent responses in the receiving field of cells .
however , it is becoming increasingly clear that for some ligands , for example , hedgehog , wnt , and fgf , other mechanisms , such as short - range signaling activation , transcriptional feedback , and cellular rearrangements , underlie morphogen function ( alexandre et al .
regulation of nodal signaling in the zebrafish embryo has long served as a paradigm for understanding how morphogens pattern tissues ( schier , 2009 ) .
nodals are secreted ligands that belong to the tgf- superfamily of growth and differentiation factors . during vertebrate development
, nodal is required for stem cell maintenance , specification of mesoderm and endoderm ( mesendoderm ) , and establishment of left - right asymmetry ( shen , 2007 ) .
recent work has also suggested that nodal signaling is reactivated in advanced cancers , where it may be important for self - renewal of cancer stem cells ( wakefield and hill , 2013 ) .
nodal ligands signal through serine / threonine kinase receptor complexes comprising two type i receptors ( acvr1ba [ taram - a ] ) , two type ii receptors ( acvr2a / b ) , and the co - receptor tdgf1 ( cripto / oep ) ( schier , 2009 , shen , 2007 ) . ligand binding activates the receptors , after which the type i receptor phosphorylates the intracellular signal transducers smad2 and smad3 , which then bind smad4 ( massagu , 2012 ) .
these smad2/3-smad4 complexes accumulate in the nucleus , where , together with transcription factors such as foxh1 , mixer , and oct4 , they regulate gene transcription ( gaarenstroom and hill , 2014 ) . at zebrafish
blastula stages , two nodal - related ligands , ndr1 ( squint ) and ndr2 ( cyclops ) , specify mesendoderm in marginal cells around the circumference of the embryo by inducing a smad2-smad4-foxh1-dependent transcriptional program ( feldman et al . , 1998 , gritsman et al . , 1999 ) .
ndr1/2 are thought to form a signaling gradient by diffusion , extending up to about ten cell tiers from the margin ( dubrulle et al . , 2015 ,
indeed , expression of presumed long - range nodal target genes such as ta ( ntla ) and fscn1a suggests low - level signaling up to ten cell tiers from the margin ( bennett et al . , 2007 ) . this appears supported by bimolecular fluorescent complementation experiments ( harvey and smith , 2009 ) .
however , other nodal target genes are expressed in up to five to six cell tiers from the margin , which coincides with nuclear accumulation of smad2-gfp fusion protein ( dubrulle et al . , 2015 ) .
importantly , other signaling pathways , such as bmp , wnt , and fgf , are also active at the margin , which can potentially co - regulate nodal target genes and thus contribute to their expression domains .
formation of the nodal signaling domain at the correct time and of appropriate dimensions is thought to be controlled by a reaction - diffusion system ( meinhardt , 2009 , schier , 2009 ) .
this model requires positive and negative feedback , which is provided by nodal - induced expression of both the ligands ndr1/2 and the antagonists lefty1 ( lft1 ) and lefty2 ( lft2 ) ( chen and shen , 2004 , cheng et al . , 2004 ) .
besides these feedback mechanisms , the model requires lft1/2 to be more diffusible than ndr1/2 ( mller et al . , 2012 , schier and talbot , 2005 ) .
these conditions are thought to allow ndr1/2 to activate signaling at the margin , whereas lft1/2 proteins would inhibit signaling in more distal cells .
overexpression studies have shown that ndr1/2 and lft1/2 can differentially diffuse and that ndr1 , but not ndr2 , can diffuse over a distance to activate signaling ( chen and schier , 2001 , chen and schier , 2002 , mller et al . , 2012 ) .
however , the importance of diffusion of endogenous ndr1/2 remains unclear , as mesendoderm can develop normally in zygotic ndr1 mutants ( dougan et al .
, 2003 , feldman et al . , 1998 , lim et al . ,
in addition to the negative feedback provided by lft1/2 , nodal signaling is regulated by the mir-430/427/302 family of micrornas ( mirnas ) ( bassett et al . , 2014 , choi et al
at blastula stages , the mir-430 family is the most abundant family of mirnas in the zebrafish .
importantly , mir-430 regulates ndr1 , lft1 , and lft2 , but not ndr2 , and this is thought to dampen nodal signaling ( choi et al . , 2007 ) .
however , to what extent mir-430s contribute to the formation of the nodal signaling domain is unknown . to develop a specific readout for endogenous nodal signaling , avoiding overexpression of any pathway components
, we generated a transgenic zebrafish nodal reporter line . using this line combined with immunofluorescence for phosphorylated smad2 ( p - smad2 )
, we show that nodal signals exclusively in cells that express ndr1/2 , up to five to six cell tiers from the margin .
this prompted us to revisit the mechanism underlying the formation of the nodal signaling domain .
our data do not support the reaction - diffusion model , but instead , we propose that nodal activates signaling in a temporal window that is defined by a mir-430-mediated delay of lft1/2 translation . in this way , temporal information is converted into spatial information in the developing embryo .
the range of activity of the nodal signaling pathway in the blastula margin has mainly been inferred from the expression of endogenous target genes , such as fscn1a and in particular ta ( ntla ) ( bennett et al .
, 2007 , gritsman et al . , 1999 ) . however , in addition to nodal , fgf signaling is also known to regulate ta expression ( griffin et al .
, 1995 , rodaway et al . , 1999 , schier and talbot , 2005 ) .
genes encoding fgf ligands , such as fgf3 and fgf8a , are expressed in the margin ( figure s1a ) and are known nodal targets ( mathieu et al . , 2004 ) , suggesting that fgf signaling at the margin of blastula - stage embryos is downstream of nodal signaling ( rodaway et al . , 1999 ) .
this is clearly demonstrated by treating embryos with the nodal inhibitor sb-505124 ( hagos and dougan , 2007 ) , which results in a near complete loss of phosphorylated erk ( p - erk ) , a readout for fgf pathway activity ( dorey and amaya , 2010 ) ( figures 1a and 1b ) .
given that both nodal and fgf signaling are active at the margin , we examined to what extent fgf signaling regulates endogenous nodal target genes , focusing on the expression of ta and fscn1a as examples of long - range genes and lft1 and lft2 as examples of short - range target genes ( bennett et al .
to inhibit fgf signaling , wild - type ( wt ) embryos were treated with the fgf receptor ( fgfr ) inhibitor su-5402 ( mohammadi et al . , 1997 ) or
were injected with mrna encoding a dominant - negative fgfr ( dnfgfr ) ( amaya et al . , 1991 )
both treatments resulted in a reduction in the size of the expression domains of ta and fscn1a in the margin of 40%50% epiboly embryos , but not of lft1 or lft2 ( figure 1d ) .
similarly , morpholinos ( mos ) against fgf3 and fgf8a resulted in a reduction of ta expression , but not of lft1 ( figure s1b ) .
qpcr on su-5402-treated 50% epiboly embryos confirmed the whole - mount in situ hybridization ( wish ) results ( figure 1e ) , and as expected , inhibition of nodal signaling by sb-505124 led to reduction in expression of all four genes ( figure 1e ) .
importantly , fgfr inhibition had no effect on c - terminal phosphorylation of smad2 ( p - smad2 ) or overall smad2 levels , demonstrating that nodal signaling is not affected by fgf signaling inhibition ( figure 1f ) . to quantitate the effect of inhibiting fgf signaling on the ta expression domain
, we performed serial sectioning on ta - stained embryos at 40% epiboly . in control embryos ,
ta is expressed in an average of about 10 cell tiers from the margin , whereas expression was reduced to six cell tiers in su-5402-treated embryos ( figures 1 g and 1h ) .
this indicated that ta expression beyond six cell tiers was due to fgf signaling and not directly dependent on nodal . in support of this idea , injection of increasing doses of fgf8a mrna into a maternal zygotic ( mz )
tdgf1 background confirmed that nodal signaling is not required for induction of ta by fgf , excluding a requirement for synergism between nodal and fgf signaling for ta expression beyond six cell tiers ( figure s1c ) .
this was further confirmed by the observation that inhibition of nodal signaling from the 16-cell stage resulted in the loss of both ta and lft1 expression , but when nodal signaling was inhibited from dome stage , only lft1 expression was severely reduced , whereas the expression of ta was unaffected ( figure s1d ) . thus ta expression is not dependent on nodal activity after fgf signaling has been initiated .
together these data demonstrate that fgf signaling regulates presumed long - range endogenous nodal target genes beyond six cell tiers . to exclusively monitor nodal signaling without inputs from other pathways
an egfp reporter gene under the control of three foxh1 and smad binding sites , termed activin response elements ( ares ) ( germain et al . , 2000 ) , was inserted into the zebrafish genome using tol2-mediated transgenesis ( tg[are : egfp ] ) ( figure 2a ) .
we chose this reporter , which we have extensively characterized in a number of cell culture and developmental contexts ( germain et al .
, 2000 , inman and hill , 2002 , randall et al . , 2004 ) , because foxh1 is the primary transcription factor required for immediate early nodal target gene expression ( pogoda et al . ,
in addition , smad2 is the predominant receptor - regulated smad during blastula stages ( figure s2a ) , and indeed mz deletion of smad2 results in a phenotype identical to mztdgf1 embryos ( dubrulle et al . , 2015 ) .
in four independent tg(are : egfp ) lines , wish for egfp mrna revealed that the reporter was activated in identical domains , excluding any effects caused by different integration sites of the transgene ( figures 2a and s2b ) .
the egfp expression domains correspond to the expression domains of nodal ligands ( schier , 2009 ) and include the embryonic margin at blastula stages , axial mesoderm during gastrulation , and the left lateral plate mesoderm during somitogenesis ( figures 2a and s3 ) .
incubation of developing tg(are : egfp ) embryos with sb-505124 resulted in a strong reduction of egfp staining ( figure 2b ) .
this was also the case when foxh1 mos were injected or when the tg(are : egfp ) embryos were bred into a tdgf1 background ( figure 2b ) .
the tg(are : egfp ) reporter is therefore specific for smad2-smad4-foxh1-mediated nodal signaling during blastula stages . to confirm the inducibility of the reporter gene , we exposed dissociated cells from tg(are : egfp ) blastula - stage embryos to increasing concentrations of recombinant nodal and measured gene expression by qpcr .
egfp expression was induced at identical nodal concentrations compared with ta , suggesting equal sensitivity of the reporter compared with this target gene ( figure 2c ) .
historically the sensitivity of nodal target genes , such as ta and noto ( flh ) , has been assessed by ectopically expressing nodal ligands in the animal pole of blastula - stage embryos and using wish to assay gene expression ( chen and schier , 2001 ) .
using nodal - coated beads in such an assay , we found that the reporter was induced in a domain of similar size to that of ta and noto ( figure 2d ) .
finally , we found no differences in the expression of downstream nodal genes in 40%50% epiboly embryos by qpcr and wish when we compared wt and tg(are : egfp ) embryos , demonstrating that the introduction of the transgene had no impact on nodal signaling ( figures s2c s2e ) . in conclusion ,
the tg(are : egfp ) zebrafish line is a specific and sensitive biosensor for early nodal signaling .
we next used the tg(are : egfp ) reporter line to determine how nodal signaling is initiated in the embryo . at the 8-cell and 1,000-cell stages ,
no egfp mrna could be detected ( figure 2e ) , indicating that egfp mrna is not maternally contributed . at sphere stage , however , signaling cells are detected as a localized cluster of egfp - positive cells ( figure 2f ) . from 30% epiboly until the onset of gastrulation , nodal signaling is detected in the entire margin , with a shallow staining gradient running from dorsal to ventral ( figure 2f ) .
as expected , the overall expression of egfp correlated well with increasing levels of p - smad2 ( figure 2 g ) .
we compared the spatial and temporal activation of nodal signaling in tg(are : egfp ) embryos with the expression of core components of the pathway ( figure s3 ) .
the genes encoding the receptor acvr1ba , the co - receptor tdgf1 , and transcription factors smad2 and foxh1 are ubiquitously expressed during blastula stages .
the activation of signaling therefore depends exclusively on ligand expression ( figure s3 ) .
the discrete signaling domain at sphere stage likely corresponds to the future dorsal side of the embryo , given the known dorsal localization of maternal ndr1 mrna ( figures s3 and s4a ) ( gore et al .
we could show that ndr1 transcripts are polyadenylated before the maternal - to - zygotic transition ( figure s4b ) , suggesting that maternal ndr1 is translated and may signal during blastula stages .
this was demonstrated by knocking down the mix - like transcription factor mxtx2 , which is required for zygotic ndr1 and ndr2 expression in the yolk syncytial layer ( ysl ) ( fan et al .
, 2007 , hong et al . , 2011 , xu et al . , 2012 ) .
tg(are : egfp ) mxtx2 morphants retained only a small , dorsal domain that expressed ndr1 , ndr2 , and egfp ( figure s4c ) .
loss of the ventrolateral expression of ndr1/2 in the blastoderm in mxtx2 morphants suggested that this ndr1/2 expression was initiated by nodal ligands secreted by the ysl and resulted from the ability of ndr1/2 to induce their own expression .
indeed , embryos incubated with the nodal inhibitor sb-505124 from the 32-cell stage exhibited complete loss of expression of ndr1/2 in the blastoderm of 40% epiboly embryos , whereas expression of ndr1/2 in the ysl was not affected ( figure s4d ) ( fan et al . , 2007 ) .
thus , maternally provided ndr1 activates nodal signaling in dorsal - most embryonic cells before the initiation of signaling in the entire margin by ndr1/2 synthesized in the ysl ( figure s4e ) .
the tg(are : egfp ) zebrafish line provides an ideal tool to investigate the dimensions of the nodal signaling domain at the margin . to initially investigate whether there was any nodal signaling beyond the ligand - expressing domain , we performed double fluorescent wish for ndr2 and egfp in 30% epiboly embryos , when the nodal signaling domain is expanding ( dubrulle et al . , 2015 , harvey and smith , 2009 ) .
although individual signaling cells were occasionally observed directly adjacent to ndr2-expressing cells , no egfp - positive cells were detected further beyond the ligand expression domain ( figure s5a ) .
to determine the extent of nodal signaling more rigorously , 40%50% epiboly tg(are : egfp ) embryos were stained for egfp , ndr1 , ndr2 , lft1 , and lft2 expression , sectioned , and quantitated ( figures 3a and 3b ) .
the expression of egfp , ndr1 , ndr2 , and lft1 was limited to an average of about five cell tiers , whereas lft2 expression was detected in only two to three cell tiers from the margin . to confirm
that these findings were not due to a lack of sensitivity of the wish , we analyzed the expression of sox3 , which is repressed by nodal signaling in the margin ( bennett et al .
significant repression was seen in up to six cell tiers from the margin ( figure 3a ) .
importantly , staining for all induced nodal target genes , including egfp , was consistently stronger in a cluster of cells directly proximal to the ysl , where signaling originates ( figure 3a ) . to corroborate the data obtained from the sections
, we used a direct readout of the nodal pathway , fluorescent immunostaining of whole - mount 50% epiboly embryos with an antibody against p - smad2 . for comparison , we also stained for p - erk .
nuclear p - smad2 staining was observed exclusively in the margin and , as expected , in a smaller domain than the p - erk staining ( figure 3c ) . to quantitate the size of the p - smad2-positive domain , we imaged the embryos at the margin , used metamorph software to generate normalized nuclear staining intensities , and measured the distance of each nucleus from the margin in control- and sb-505124-treated embryos .
p - smad2 staining above background was found up to five to six cell tiers ( 8090 m ) from the margin in a steep gradient , which was abolished in sb-505124-treated embryos ( figures 3d , 3e , and s5b s5d ) .
the staining was strongest in the nuclei of cells nearest the ysl . taken together with our observations from the tg(are : egfp ) reporter line , these data demonstrate that nodal signaling occurs in five to six cell tiers from the margin , closely mirroring the expression of the ligands .
our data demonstrate that the nodal ligands and antagonists are co - expressed at the margin of late blastula - stage embryos , and moreover , nodal activity is restricted to these cells .
first , if the ligands and antagonists are co - expressed , how does signaling occur at all ?
second , given that all cells are competent to signal at blastula stages and nodal signaling induces the expression of the ligands , why does signaling not spread throughout the embryo ? to address these questions , we first determined to what extent lft1/2 regulate nodal signaling in early and late blastula - stage embryos .
the lft1/2 proteins are known to inhibit signaling by sequestering tdgf1 and possibly also nodal itself ( chen and shen , 2004 , cheng et al . , 2004 ) . as expected ,
injection of lft1/2 mos led to an expansion of the nodal signaling domain at 50% epiboly , which was confirmed by qpcr ( figures 4a and 4b ) . at dome stage , however , there was no increase in the size of the nodal signaling domain , suggesting that lft1/2 do not regulate nodal signaling at early stages .
although there is an offset in the appearance of ndr1/2 and lft1/2 mrna due to the presence of maternal ndr1 transcripts , the lack of an early role for lft1/2 is not explained by an absence of lft1/2 mrna at sphere and dome stages ( figures 4c and s3 ) .
we hypothesized therefore that a delay in translation of the lft1/2 proteins could account for the inability of lft1/2 to regulate nodal signaling at dome stage .
both lft1 and lft2 transcripts were readily polyadenylated by sphere stage ( figure 4d ) , suggesting that lack of polyadenylation could not account for any delay in lft1/2 translation . to measure endogenous protein levels directly ,
we raised polyclonal antibodies for lft1 and lft2 and thoroughly characterized them ( figure s6 ) .
although both antibodies recognized their corresponding target protein when overexpressed ( figure s6b ) , only the lft1 antibody was able to detect endogenous protein , so we focused on this family member .
the major band detected by western blot corresponding to endogenous lft1 migrated by sds - page with a molecular weight of 40 kd ( figure s6c ) .
mutation analysis indicated that this product arose from cleavage at the first furin cleavage site ( marked as c1 in figure s6a ) , and we could demonstrate that this product was active ( figures s6d s6f ) .
the same 40 kd band was also detected in embryos injected with ndr1 mrna ( figure s6
g ) . levels of endogenous lft1 were barely detectable by western blotting at dome stage and 30% epiboly but increased at 50% epiboly ( figures 4e and 4f ) .
thus , endogenous lft1 protein levels remain low until 50% epiboly , despite readily detectable mrna levels at all these time points .
this suggested that the lack of an early role for lft1/2 could be due to low protein abundance and led us to hypothesize that repressed lft1/2 translation creates a window of opportunity for nodal signaling to become established . during blastula stages , the mir-430 family of mirnas
have been reported to block translation , without affecting polyadenylation ( bazzini et al .
we reasoned therefore that the activity of mir-430 could be responsible for the repressed translation of lft1/2 proteins to create a temporal window for nodal to activate signaling .
mir-430 pri - mirna is expressed in the nuclei of all cells in the blastoderm , immediately after activation of zygotic transcription ( figure 5a ) .
we also confirmed a ubiquitous expression pattern for mature mir-430a and mir-430b at 50% epiboly ( figure 5b ) and demonstrated that mature mir-430a and mir-430b are directly processed upon expression ( figure 5c ) .
mir-430c was not detected by either wish or northern blotting , but rna sequencing data demonstrated that mir-430c is much less abundant than mir-430a and mir-430b ( unpublished data ) .
thus , the mir-430 family is abundant , ubiquitously expressed , and readily processed during mid to late blastula stages .
to determine the role of mir-430 in the regulation of lft1/2 protein translation , we designed three mos that prevented processing of mature mir-430a , mir-430b , and mir-430c .
injection of these mos into one - cell - stage embryos resulted in a phenotype resembling mz dicer mutants at 22 hpf ( figure s7a ) ( giraldez et al . , 2005 ) .
furthermore , they abolished mir-430a staining at 50% epiboly and reduced mir-430a , mir-430b , and mir-430c expression , as determined by qpcr , by 89% ( figures s7b and s7c ) .
co - injection of mir-430 mos with a gfp reporter containing either three mir-430 binding sites or a gfp reporter with the lft2 3utr , which contains a single mir-430 binding site , resulted in increased translation compared to control mos ( figure s7d ) .
we next injected equal amounts of control or mir-430 mos and performed western blotting for p - smad2 and lft1 at several blastula stages . in control
mo - injected embryos , lft1 protein was not detectable until 30% epiboly and increased at 50% epiboly , as observed for lft1 protein expression in wt embryos ( compare figure 4e with figure 5d ) .
importantly , this was accompanied by a gradual increase of p - smad2 over time .
in contrast , injection of mir-430 mos led to premature translation of lft1 from dome stage , and this coincided with lower overall accumulation of p - smad2 ( figure 5d ) .
interestingly , the level of lft1 protein in dome - stage mir-430 morphants was similar to the maximal level of lft1 protein measured at 50% epiboly in control mo - injected embryos , suggesting that there may be a threshold level of lft1 that is inhibitory . to determine whether reduced signaling in the mir-430 morphants was due to premature translation of lft1/2 and to investigate the spatial consequences for nodal signaling of the loss of mir-430 , we injected mir-430 mos and/or lft1/2 mos into tg(are : egfp ) embryos and assayed nodal activity ( egfp ) and ndr1 levels at 30% epiboly .
injection of lft1/2 mos alone resulted in a modest increase in egfp and ndr1 staining ( figure 5e ) , whereas injection of mir-430 mos led to a reduction in egfp staining in the blastoderm , consistent with the inhibition of the nodal signaling pathway we observed using p - smad2 levels as a readout ( figure 5d ) .
when lft1/2 mos and mir-430 mos were co - injected , signaling was activated in the entire blastoderm , and this was accompanied by a similar expansion of ndr1 expression ( figure 5e ) . importantly , this spreading of egfp staining in lft1/2 and mir-430 mo - co - injected embryos was due to nodal signaling , because it was completely blocked by incubating double morphants with sb-505124 ( figure 5f ) .
the further spreading of signaling following combined knockdown of lft1/2 and mir-430 is readily explained by the regulation of ndr1 translation by mir-430 in the absence of lft1/2 ( see figure 7a ) ( choi et al . , 2007 ) .
together , these experiments demonstrate that mir-430 delays lft1/2 translation to create a temporal window for nodal to activate signaling .
our data demonstrate that a temporal window for nodal signaling activation determines the size of the nodal signaling domain and predict that once lft1/2 levels reach a certain threshold , nodal signaling is unable to spread to adjacent cells .
we therefore tested if blocking signaling activation by recombinant mouse lefty1 ( mlefty1 ) is dose dependent in dissociated embryonic cells .
a 5-fold excess ( calculated by mass ) of mlefty1 over human recombinant nodal led to a near complete inhibition of signaling activation as read out by western blotting for p - smad2 and endogenous zebrafish lft1 , which monitors the transcriptional output of the pathway ( figure 6a ) .
thus , lft1/2 proteins can reach an inhibitory concentration at which signaling can no longer be activated .
next we determined if duration of exposure to nodal directly corresponds to increasing levels of signaling .
when blastula - stage cells were exposed to 50 ng / ml nodal and then inhibited with a blocking concentration of 500 ng / ml mlefty1 at different time points , p - smad2 and endogenous lft1 levels were indeed proportional to the duration of ligand exposure ( figure 6b ) .
finally , we investigated how rapidly signaling is blocked when lft1/2 levels reach inhibitory concentrations .
this is a crucial issue , as we observe that nodal signaling in vivo ( as read out by egfp , ndr1 , ndr2 , lft1 , and lft2 ) is sustained in the margin for several hours after lft1 levels reach an inhibitory concentration at around 50% epiboly ( figure s3 )
. dissociated embryonic cells were therefore exposed to nodal for 1 hr , and then signaling was inhibited by addition of mlefty1 for 2 hr . we observed that p - smad2 levels decreased slowly , compared with the rapid termination of signaling with sb-505124 ( figure 6c ) .
this demonstrated that although signal activation is blocked by lefty , nodal signaling is sustained for some time , presumably because of continued signaling from internalized receptor complexes in early endosomes ( jullien and gurdon , 2005 , vizn et al . , 2013 ) .
in conclusion , our data show that activation of nodal signaling in blastula - stage cells can occur until lft1/2 levels reach inhibitory concentrations .
moreover , the levels of p - smad2 , and as a result transcription , are proportional to the duration of signal activation .
this can be maintained for some time after inhibitory lft1/2 concentrations are reached , while no new signaling is activated .
here we describe a specific and sensitive nodal reporter line that has enabled us to visualize endogenous nodal signaling in developing zebrafish embryos , without overexpression of any pathway components .
we show that signaling is initiated on the dorsal side because of maternally provided ndr1 .
ventral and lateral signaling arises as a result of ndr1/2 expression in the ysl , which then spreads toward the animal pole as a result of autoregulation .
nodal signaling in the margin reaches a maximum of six cell tiers , which we demonstrate by p - smad2 immunostaining and reporter activity .
we find no evidence of signaling beyond the cells that express the ligand , and moreover , these same cells additionally express the nodal antagonists lft1/2 . spreading of presumed long - range nodal target genes , such as ta , that are activated beyond the ligand expression domain ,
our data support a model whereby a temporal window for nodal signaling activation dictates the dimensions of the nodal signaling domain ( figure 7 ) .
thus , temporal information is translated into spatial information in the developing embryo . the crucial determinant of the temporal window is the delayed translation of the lft1/2 proteins , which is mediated by mir-430 . in addition , maternally provided ndr1 transcripts and the production of ndr1/2 by the ysl allow nodal signaling to be initiated in the blastoderm , before transcription of lft1/2 ( figure 7c ) .
because of positive feedback , the blastoderm cells produce more ndr1/2 , while lft1/2 levels remain relatively low because of the ubiquitous synthesis of mir-430 . in these conditions ,
nodal signaling can be activated in neighboring cells until extracellular lft1/2 levels reach inhibitory concentrations .
therefore , the duration for which lft1/2 levels are repressed dictates the size of the nodal signaling domain . how the repressive action of mir-430 is lifted at 50% epiboly to allow lft1/2 translation is not yet known and requires further investigation .
although inhibitory lft1/2 levels prevent further activation of signaling , and hence additional spreading of nodal signaling , cells already responding to nodal will continue to signal for several hours , because this occurs from internalized receptors that are refractory to lft1/2 inhibition .
a consequence of our proposed mechanism is that cells directly adjacent to the ysl activate nodal signaling for the longest duration .
this likely explains the more intense p - smad2 staining in these cells relative to those further from the margin .
we therefore propose that nodal signaling at the margin at blastula stages is dictated by an interplay among ligand , ligand antagonist , and a mirna , with a differential in timing between ligand and antagonist production being the key determining factor .
the size and shape of the nodal signaling gradient had previously been thought to be regulated by a reaction - diffusion system ( meinhardt , 2009 , mller et al . , 2012 , schier , 2009 ) .
here we propose an alternative mechanism whereby the size of the domain is dictated by the delay in lefty translation .
although the nodal / lefty ligand / antagonist pair has many features of a reaction - diffusion system , we have uncovered one aspect that is incompatible . in reaction - diffusion models , which were originally conceived as
pattern - forming chemical reactions , a homogeneous distribution of activator and inhibitor are unstable , and a local elevation of activator initiates formation of a gradient ( meinhardt , 2009 ) .
integral to this model is the ability of the diffusing antagonist to immediately inhibit activator function at a distance . for nodal and lefty
, this can not happen , because once nodal signaling is activated it occurs from internalized receptors and is therefore insensitive to lefty inhibition , except over prolonged time frames .
our data suggest that spatially graded activity of the nodal signaling pathway is mainly the result of different durations of exposure to ndr1/2 over time , as opposed to exposure to different concentrations .
we observe two graded signaling domains in tg(are : egfp ) embryos that are both explained by timing of ligand exposure . during blastula stages , a shallow signaling gradient runs from dorsal to ventral .
this is readily explained by the fact that dorsal cells are exposed to nodal for a longer period than ventrolateral cells , because maternally provided ndr1 signals dorsally before ndr1/2 produced in the ysl induces signaling in the blastoderm margin .
in addition , we found a clear vegetal - to - animal gradient within the ligand - expression domain using p - smad2 immunostaining in late blastula - stage embryos , and we also observed that cells directly adjacent to the ysl expressed higher levels of all nodal target genes , including the egfp reporter gene , reflecting higher levels of signaling .
again , cells directly adjacent to the ysl are exposed to ndr1/2 for the longest period of time .
the importance of duration of exposure is further supported by our ex vivo experiments with dissociated embryonic cells .
the long - term functional consequences of this were demonstrated in previously published work , which linked cell fates to the duration of exposure to nodal signals ( hagos and dougan , 2007 ) .
thus , our model explains how concentration and duration of signaling can be translated into positional information .
finally , the importance of timing of signaling activation also rationalizes the normal development of ndr1 mutants ( feldman et al .
, 1998 , heisenberg and nsslein - volhard , 1997 , lim et al . , 2013 ) .
this is explained by the fact that ndr2 compensates for the lack of ndr1 , but its expression is delayed , because there is no maternal ndr2 . from the sectioning of tg(are : egfp ) embryos and immunostaining for p - smad2 , it is clear that smad2-smad4-foxh1-dependent nodal signaling is confined to 56 cell tiers from the margin , which could imply that all mesendodermal cells arise from this domain .
this finding seems to contradict lineage - tracing studies that show that some mesodermal precursors are located up to 12 cell tiers away from the margin ( e.g. , see dougan et al .
although these lineage - tracing studies have provided valuable insight into the overall spatial distribution of mesendoderm precursors , they can not themselves determine the extent of nodal signaling .
this is because although nodal signaling is required for mesendoderm formation , not all cells that become mesendoderm have necessarily experienced nodal signaling directly .
our understanding of how morphogens activate graded signaling in tissues has been dominated by the pre - molecular era assumption that secreted ligands diffuse from a source to form concentration gradients , and this assumption has naturally progressed into the formulation of models that include diffusion as a major determinant in patterning by morphogens .
although in some contexts , such as the establishment of left - right asymmetry , nodal can act at long range ( shiratori and hamada , 2014 ) , the work presented here shows that the formation of the nodal signaling domain at the blastula margin is explained by short - range signaling activation , signaling dynamics , and transcriptional / translational regulation .
human nodal ( 3218-nd / cf ; r&d ) was dissolved in 4 mm hcl at 100 g / ml , aliquoted in non - stick tubes , and used at 40 ng / ml ( unless stated otherwise ) without freeze - thawing .
recombinant mlefty1 was dissolved according to the manufacturer s instructions ( 994-lf / cf ; r&d ) .
the inhibitors sb-505124 ( 3263 ; tocris bioscience ) and su-5402 ( 572631 ; calbiochem ) were dissolved in dmso and used in embryos at 50 and 10 m respectively . in dissociated embryonic cells ,
up to 1,000 embryos were dechorionated using 2 mg / ml pronase ( 11459643001 ; roche ) in 10 ml e3 medium .
the dechorionated embryos were washed extensively in e3 medium and once with calcium - free ringers buffer to remove the pronase ( link et al . , 2006 ) .
the embryos were manually disrupted in calcium - free ringers buffer using a p200 pipette in 6 cm bacterial dishes , collected by centrifugation at 1,000 g for 5 min , and then resuspended at 50 embryos / ml in leibovitz s l15 medium ( 11415 - 064 ; gibco ) supplemented with 3% fetal bovine serum ( fbs ) .
cells were plated in 24-well tissue culture plates coated with poly - l lysine ( p4707 ; sigma ) and allowed to attach for 30 min .
embryos were fixed in 4% paraformaldehyde in pbs overnight , dehydrated to 100% methanol , and stored at 20c until processing . for whole - mount immunofluorescence
, embryos were rehydrated to pbs and incubated in cold acetone at 20c for 20 min . blocking and
antibody incubations were performed in 10% fbs and 1% triton x-100 in pbs , and washes were performed in pbs/1% triton .
the following primary antibodies were used : -p - smad2/3 ( 8828 ; cell signaling technology ) and -p - erk ( m8159 ; sigma ) . note that because of the lack of smad3 at blastula stages , we solely detect p - smad2 with the -p - smad2/3 antibody .
dapi was used to stain nuclei , and images were acquired on a zeiss lsm 780 confocal microscope . for quantification of p - smad2 intensity in deep cells , metamorph software ( molecular devices ) was used to generate p - smad2 to dapi ratios from at least three single optical slices per embryo , in three individual dmso or sb-505124 treated , 50% epiboly embryos .
care was taken not to select mitotic or overlapping nuclei or nuclei from the enveloping layer . to measure the distance of a nucleus to the margin
, a line was drawn laterally at the vegetal edge of the margin , and the distance to the nucleus perpendicular to the margin was measured using the metamorph software . to normalize the staining intensity for each embryo ,
the average ratio of an area further than 11 cell tiers from the margin ( > 165 m ) was subtracted from each measurement .
the data were divided in 15 m bins , representing the average size of a deep cell at 50% epiboly ( unpublished data ; dubrulle et al . ,
the averages of the binned data for each cell tier , comparing dmso and sb-505124 treatment , were used for testing for statistical significant differences using paired t tests with a 95% confidence interval . all the zebrafish work was carried out under a uk home office license under the animals ( scientific procedures ) act 1986 .
the license underwent full ethical review and approval by the cancer research uk london research institute animal ethics committee .
, c.h . , and m .- c.r . performed the experiments and analyzed the data with the help of r.k.s . | summarymorphogen signaling is critical for the growth and patterning of tissues in embryos and adults , but how morphogen signaling gradients are generated in tissues remains controversial .
the morphogen nodal was proposed to form a long - range signaling gradient via a reaction - diffusion system , on the basis of differential diffusion rates of nodal and its antagonist lefty .
here we use a specific zebrafish nodal biosensor combined with immunofluorescence for phosphorylated smad2 to demonstrate that endogenous nodal is unlikely to diffuse over a long range . instead , short - range nodal signaling activation in a temporal window is sufficient to determine the dimensions of the nodal signaling domain .
the size of this temporal window is set by the differentially timed production of nodal and lefty , which arises mainly from repression of lefty translation by the microrna mir-430 .
thus , temporal information is transformed into spatial information to define the dimensions of the nodal signaling domain and , consequently , to specify mesendoderm . |
stickler syndrome alternately known as hereditary arthro - ophthalmopathy is an inherited disorder of collagen formation manifesting itself through a spectrum of ocular , orofacial , auditory , and musculoskeletal abnormalities.1,2 the disease is relatively uncommon , with a reported incidence of one in 7,5009,000 births , and presents with highly variable signs and symptoms in affected individuals.3 this diversity of phenotypes reflects the heterogeneous mutations affecting the formation of types ii , ix , and xi collagen , which are integral to the structure and function of the vitreous , cartilage , and other connective tissues.4,5 specific ocular manifestations include high myopia , optically empty vitreous , open - angle glaucoma ( from dysgenesis of the anterior chamber drainage angle ) , cortical cataract , and perivascular retinal lattice degeneration with strong vitreous adhesions along the borders of these lesions.6 these vitreoretinal changes predispose patients to the development of giant retinal tears ( grts ) and associated rhegmatogenous retinal detachments ( rrd ) relatively early in life , often in the second and third decades of life.4,6,7 stickler syndrome is the most common inherited cause of rrd , in spite of its low incidence overall.8 given the low incidence of stickler syndrome , long - term data on surgical outcomes in patients with stickler syndrome are few : only two published studies have previously detailed the surgical treatment and outcomes of retinal detachment ( rd ) repair in patients with stickler syndrome.9,10 we reviewed the long - term anatomical and visual outcomes of rd in our cohort of patients with stickler syndrome who underwent surgical repair .
this study is a retrospective , single - center , interventional , case series of patients with stickler syndrome undergoing surgery for rd .
patients were screened via billing codes , surgical logs , and genetic testing and were seen during the period from january 1 , 2009 , through december 31 , 2014 .
patient records were reviewed to confirm a diagnosis of stickler syndrome , and data collection was completed , as outlined later .
patients were excluded if they did not have a confirmed diagnosis of stickler syndrome , if there were confounding causes for rrd ( eg , ocular trauma ) or confounding reasons for severe vision loss ( eg , end - stage glaucoma ) , or if they did not undergo surgery primarily for rd .
the study was approved by the western institutional review board , and it complied with the health insurance portability and accountability act of 1996 and adhered to the tenets of the declaration of helsinki . the requirement to obtain informed consent from patients was waived due to the nature of the study .
data collection included demographic information , details of surgical interventions , and multimodal retinal imaging including color fundus photography , fluorescein angiography , and optical coherence tomography .
best corrected visual acuity ( bcva ) and anatomical outcomes were recorded pre - and postoperatively .
snellen visual acuities were converted to the logarithm of the minimum angle of resolution units for statistical analyses .
nonparametric paired significance testing was performed using the wilcoxon signed - rank test ; statistical tests were two - tailed , and significance was defined as p<0.05 .
stata version 9.0 ( statacorp lp , college station , tx , usa ) was used for statistical analyses .
sixteen eyes from 13 patients with stickler syndrome met the inclusion criteria , with three patients ( 23% ) undergoing bilateral surgeries for rd repair . the average age at the time of initial diagnosis with rrd was 10.4 years ( range : 1 month22 years ) , and the mean follow - up duration was 94 months ( range : 5313 months ) .
mean visual acuity ( va ) at the time of presentation was 1.66 ( sd 0.86 ; snellen equivalent : 20/914 ) , which slightly improved at final evaluation to 1.60 ( sd 1.01 ; snellen equivalent : 20/796 ; p=0.81 ) .
improvement in va was seen in five eyes ( 31% ) , while five ( 31% ) remained stable and six ( 37.5% ) experienced a decline .
three eyes ( 18.8% ) had single retinal breaks , five eyes ( 31.3% ) had multiple breaks , and the remaining eight ( 50% ) had grts with at least 3 clock - hours of peripheral retinal involvement .
the mean number of vitreoretinal surgeries required for retinal reattachment was 3.1 ( range : 113 ) . in eyes requiring multiple surgical interventions ( 13 of 16 eyes , 81.3% ) ,
the mean interval prior to redetachment ( after the primary intervention ) was 4.8 months ( range : 4 days36 months ) .
six retinas ( 37% ) were attached without silicone oil tamponade , while the remaining ten retinas ( 63% ) were attached utilizing silicone oil tamponade .
stickler syndrome , though rare , presents certain diagnostic and therapeutic challenges to the vitreoretinal surgeon .
several factors may contribute to the complexity of rrd repair in these patients , including the presence of multiple retinal tears , grts , the young age of presentation , and the abnormal vitreous syneresis seen in patients with stickler syndrome .
this difficulty is evident in the average number of surgeries until complete rrd repair in these patients , which was shown to be 3.1 in our study . given these confounding factors , however , all 16 eyes in our study achieved successful retinal reattachment , with a mean postoperative follow - up to almost 8 years . in our series , patients with poorer presenting va were more likely to require silicone oil tamponade for successful reattachment .
anatomical success in the presence of silicone oil tamponade is consistent with previous studies , which also showed anatomical success when silicone oil tamponade was utilized.9,10 in spite of successful anatomical outcomes , visual outcomes remain modest , improving only slightly from va at presentation : in our cohort , patients with rrd presented with an average best corrected visual acuity of 20/914 ( snellen equivalent ) . after an average of 3.1 surgeries ,
mean best corrected visual acuity only improved to 20/796 though this improvement did not achieve statistical significance ( p=0.81 ) and the va outcomes were widely variable , ranging from 20/20 to hand motions . of note , presenting va bore prognostic significance , as it showed strong correlation with the postoperative va at final follow - up ( p<0.001 ) .
the variability of va outcomes after surgical intervention for rd in our cohort is similar to those va results reported by abeysiri et al,9 where 33% of patients had improved va , 40% maintained va , and 27% experienced a decrease in va , and alshahrani et al,10 where 64.29% of patients had improved va while 28.57% maintained va .
most eyes ( 81.3% ) in our study required multiple surgeries to successfully achieve retinal reattachment .
the five eyes demonstrating the greatest improvements in va postoperatively required , on average , approximately five surgeries before retinal reattachment .
when surgical treatments are performed to repair these difficult and complex rds , our series demonstrated that initial surgery with scleral buckle , vitrectomy , and combined vitrectomy and scleral buckle procedures all yielded equally successful anatomical outcomes with 100% retinal reattachment .
these results contrast to those of previous studies , one of which showed superior anatomical outcomes with vitrectomy , while the other suggested combined vitrectomy and scleral buckle procedure with silicone oil injection to be the treatment of choice for treating rd in patients with stickler syndrome.9,10 our data overall underscore a few critical points for stickler syndrome patients with rd : first , the 75% rate of proliferative vitreoretinopathy seen in our study , as well as the average of 3.1 surgeries required to repair these rds , highlights the complicated nature of surgical therapy for these patients .
second , in our cohort , patients presented with rrd at a mean age of 10.5-years old , which is a younger age compared to previous series.9,10 finally , given this younger age of presentation , as well as the challenges of surgery and the modest visual gains , these data further reinforce the importance of early diagnosis and prophylactic therapy in patients with stickler syndrome .
previous studies have demonstrated that prophylactic cryotherapy markedly reduces the risk of rds in stickler syndrome patients.11,12 recognition of patients with a stickler ocular phenotype by primary care doctors , ophthalmologists , and other health care providers is essential for preventing the severe retinal complications in these patients
. limitations of our study include its retrospective design and the small sample size studied a frequent limitation of reviews of stickler syndrome .
this small sample size inevitably magnifies any diagnostic or therapeutic biases in our cohort , and significant caution is warranted in drawing conclusions from subgroup analyses .
however , as our data tended to corroborate those of previous publications , the conclusions drawn from the overall cohort are likely to be sound .
our study affirms the feasibility of successful retinal reattachment in cases of rrd in stickler syndrome , usually after multiple vitreoretinal surgeries .
however , va outcomes are highly variable and show only a small improvement from presenting va in long - term follow - up overall .
complicated peripheral retinal pathology , high rates of grt , high rates of proliferative vitreoretinopathy , and frequent redetachment requiring additional surgeries are the norm in this cohort , and early diagnosis ( within the first decade of life ) should be the goal for these patients . | purposethe aim of the study was to present the long - term anatomical and visual outcomes of retinal detachment repair in patients with stickler syndrome.patients and methodsthis study is a retrospective , interventional , consecutive case series of patients with stickler syndrome undergoing retinal reattachment surgery from 2009 to 2014 at the associated retinal consultants , william beaumont hospital.resultssixteen eyes from 13 patients were identified .
patients underwent a mean of 3.1 surgical interventions ( range : 113 ) with a mean postoperative follow - up of 94 months ( range : 5313 months ) .
twelve eyes ( 75% ) developed proliferative vitreoretinopathy .
retinal reattachment was achieved in 100% of eyes , with ten eyes ( 63% ) requiring silicone oil tamponade at final follow - up .
mean preoperative visual acuity ( va ) was 20/914 , which improved to 20/796 at final follow - up ( p=0.81 ) .
there was a significant correlation between presenting and final va ( p<0.001 ) , and patients with poorer presenting va were more likely to require silicone oil tamponade at final follow - up ( p=0.04).conclusionrepair of retinal detachment in patients with stickler syndrome often requires multiple surgeries , and visual outcomes are variable .
presenting va is significantly predictive of long - term va outcomes . |
role of the p53 protein in regulation of cell cycle , cell growth and maintenance of genomic stability has been largely described .
numerous alterations in p53 lead to neoplasmatic transformation . a rare familial li - fraumeni syndrome was associated with germline mutations in this gene . in families with li - fraumeni syndrome most
commonly observed neoplasms are : soft tissue sarcomas , osteosarcomas , brain tumors , breast cancer , leukemias , and adrenocortical tumors , but not ovarian cancer however , over 200 epidemiologic studies evaluated ovarian cancer risk associated with snp variants in tp53 . among them the tp53 215 g > c ( rs1042522 , p.pro72arg ) is one of the most commonly disputed .
the results differ between populations with little or no evidence for the risk of cancer for opposite pro or arg alleles [ 28 ] .
similar thus inconclusive results are observed in ovarian cancer [ 911 ] and thereby reproduce previous findings .
these equivocal data may suggest that the tp53 rs1042522 does not influence carcinogensis directly , but might be linked to another pathogenic alteration responsible for higher risk of ovarian cancer .
one of the candidate gene is the wrap53 ( also denoted as wdr79 and tcab1 ) located on chromosome 17p13 which partly overlaps the tp53 and interacts with the 5utr of p53 .
wrap53 is required for control and induction of p53 upon dna damage to maintain normal levels of p53 in the cell preventing its deprivation which may then lead to a cancer the identification of wrap53 as a regulator of p53 shows that dysfunction of wrap53 itself may be a separate cause leading to a cancer [ 13 , 14]recent reports suggested that common variations in wrap53 may independently increase risk of estrogen receptor negative breast cancers and correlates with poor prognosis of head and neck cancer .
aim of this study was the analysis of association between snps located in wrap53tp53 region : rs1042522 , rs2287497 , rs2287498 and ovarian cancer risk in the polish population .
the studied group consisted of 626 ovarian cancer patients from the registry of international hereditary cancer centre in szczecin , poland , who were diagnosed in years 20032009 in cooperating oncology centers in szczecin and pozna .
the control population consisted of 1045 healthy women who visited their family doctors in the area of szczecin .
mean age and age structure did not differ significantly between cases and controls ( age range 2679 vs 2779 , mean age 55.5 vs 56 respectively ) .
the study protocol was approved by the institutional review board of the pomeranian medical university , the approval was obtained from all cooperating institutions , and signed informed consent for genetic testing was obtained from all participants .
10 ml of peripheral blood samples for dna extraction were acquired from cases and controls .
all samples were genotyped with use of simple probes on roche lightcycler 480 in 384 well plates in 10 l pcr reaction using 1:10 dilution of lightcycler 480 genotyping master , 0.5 m of excess primer , 0.05 m of limiting primer , 0.6 m of probe and ~20 ng of dna according to roche applied sciences genotyping master mix instructions .
all statistical calculations were performed using the common homozygous of each snp as the reference value .
each of the snps was evaluated individually per genotype by calculating odds ratio ( or ) , 95 % confidence interval ( 95 % ci ) , p values using a chi test .
genotype distributions of all three examined snps in control population were in accordance with hardy weinberg equilibrium : rs1042522 ( maf = 0.28 , hwe = 0.19 ) ; rs2287497 ( maf = 0.12 , hwe = 0.65 ) , rs2287498 ( maf = 0.10 , hwe = 0.57 ) .
the distribution of the genotypes among ovarian cancer samples revealed an association of studied snps with ovarian cancer risk .
rs1042522 cc homozygotes were more frequent in cases when compared to controls ( or = 1.46 ) ( table 1 ) .
similar association was observed for wrap53 rs2287497 and rs2287498 where tt homozygotes and aa homozygotes , were more frequent among cases than controls ( or = 1.95 , 2.65 , respectively ) ( table 1 ) .
all the results were statistically significant.table 1distribution of genotypes among ovarian cancerssitecasescontrolsorpci 95 % n(%)n(%)rs10425225cc599.4726.91.460.031.002.11cg26542.343641.71.080.250.881.33gg30248.253751.41.001.00rs2287497tt203.2171.61.950.031.013.76tg11818.922021.10.890.190.721.18cc48878.080877.31.001.00rs2287498aa193.0121.22.650.011.275.50ga9915.818417.60.900.240.691.18gg50881.284981.21.001.00 distribution of genotypes among ovarian cancers a combined analysis of examined three snps showed an even stronger association with ovarian cancer risk for cases with three rare homozygotes cc + tt + aa versus wild type homozygotes gg + cc + gg ( or = 4.76 p = 0.0006 ci 95 % 1.84-12.32 ) .
however , detailed studies around this region will be needed to clarify the observed association of that specific homozygosity in other populations .
the ambiguous associations found for rs1042522 with little or no evidence for the risk of ovarian cancer were previously described in different populations , similarly neither tp53 germline mutations nor li - fraumeni syndrome was proven to be associated with an increased risk of ovarian cancer .
hence the hypothesis that an evaluation of snps located in close proximity to tp53 may reveal that rs1042522 is just a marker of other pathogenic alleles .
wrap53 is one of the candidate flanking genes to tp53 located on antisense strand to tp53 .
an analysis of the literature revealed studies involving genotyping of 23 snps in the tp53 region including some variants outside the tp53 coding sequence , among them snps located in fact within wrap53 . previously published multicenter pooled data suggested an association between rs2287497 and rs2287498 with serous invasive ovarian cancers , but the results were inconsistent between analyzed populations [ 10 , 11 ] .
rs2287498 is a synonymous amino acid change in exon 2 ( f150f ) of wrap53 gene , expected to affect function at a splice site , rs2287497 is an intronic change , however supportive of an association .
the study of schildkraut included a small number of polish ovarian cancer patients , however narrowed to serous ovarian cancer cases only , because of the a priori belief that tp53 variants might be more closely related to serous cancers .
schildkraut findings showed a trend consistent with our results , but due to relatively smaller groups ( 118 ovarian cancers vs. 618 controls ) the scores were not statistically significant leaving the case open .
our results provide the evidence for an association between snps both in tp53 and wrap53 and a risk of invasive ovarian cancer in poland .
our results might be a suggestive evidence that specific homozygosity of several snps in tp53wrap53 region is significantly overrepresented in ovarian cancer patients .
the observations suggest a stronger association of both rs2287497 and rs2287498 ( wrap53 variants ) than of rs1042522 ( tp53 variant ) and lead to a conclusion that snps in wrap53 is more strongly associated with an ovarian cancer risk than rs1042522 in tp53 .
it is possible that other variants of wrap53 , not selected for the evaluation , are also related to the disease . | among many alterations within the tp53 gene the rs1042522 ( c72 g , p.pro72arg ) has been associated with numerous cancers , however the results differ between populations for opposite pro or arg alleles .
similar thus inconclusive results are observed in ovarian cancer , which may suggest that the rs1042522 does not influence ovarian carcinogensis directly , but might be linked to another pathogenic alteration .
wrap53 which overlaps the tp53 is required to maintain normal levels of p53 upon dna damage , but also when altered may independently increase the risk of cancer . to evaluate the association between three snps located in wrap53tp53 region : rs1042522 , rs2287497 , rs2287498 and ovarian cancer risk in polish population we genotyped 626 cases and 1,045 healthy controls .
our results provide the evidence for an association between studied snps and a risk of invasive ovarian cancer in poland .
we found that cc homozygotes in rs1042522 were more frequent in cancers when compared to controls ( or = 1.46 , p = 0.03 ) .
similarly in wrap53 both tt homozygotes in rs2287497 ( or = 1.95 , p = 0.03 ) and aa homozygotes in rs2287498 ( or = 2.65 , p = 0,01 ) were more frequent among cases than healthy individuals .
there is also a suggestive evidence that specific homozygosity of studied snps in tp53wrap53 region is significantly overrepresented in ovarian cancer patients . in conclusion snps in wrap53 ( rs2287497 and rs2287498 )
have stronger association with an ovarian cancer risk than rs1042522 in tp53 . |
in modern society , chemicals are indispensable for everyday human life . numerous chemicals are used to maintain and improve the quality of life , including cosmetics , toiletries , detergents , air fresheners , agrochemicals such as pesticides and fertilizers , disinfectants , sterilizers , preservatives and industrial solvents .
chemicals are also employed in the manufacture of various product - comprising ingredients or parts like coatings / paints , photo - resistant treatments , batteries , automobiles , packaging and many more uncountable uses .
one recent report estimated that as many as 80000 to 120000 chemicals are currently in use worldwide , with an additional 2000 chemicals newly introduced each year .
in korea , approximately 44000 chemicals are known to be in use and about 300 new chemicals are newly marketed annually .
accordingly , exposure to humans and the environment is unavoidable , which can be accompanied by adverse effects to human health and/or the environment . the potential danger of chemical exposure and its devastating outcomes has been strikingly exemplified by the recent tragic accident surrounding oligo(2-(2-ethoxy)ethoxyethyl guanidine chloride / polyhexamethyleneguanidine , which were inadvertently used to sterilize humidifiers .
these chemicals , which were originally developed as carpet sterilizers , when used in humidifiers , resulted in high doses via inhalational exposure and unanticipated lung fibrosis , which cost 701 innocent lives , mostly newborn babies and nursing mothers . to better protect humans and the environment against exposure to toxic chemicals , the act on the registration and evaluation of chemicals ( k - reach ) , was enacted and has been in force since 2015 , mandating the registration of a chemical before its release onto the market with relevant information on human health and environmental hazards .
k - reach is the korean version of the european registration , evaluation , authorization and restriction of chemicals ( eu - reach ) regulation .
k - reach originally aimed to manage the risk stemming from chemical exposure by providing relevant information on intrinsic hazards and guidance regarding safe use , such as by providing proper classification and labeling . in this respect , it is aligned with the united nation ( un ) globally harmonized system of classification and labeling of chemicals ( ghs ) .
the un ghs hazard class system categorizes chemicals according to its level of health hazard as an axis and toxic outcomes or endpoints as another .
accordingly , the effects of chemicals on the respective toxic endpoints is evaluated to obtain safety data so the chemical can be properly classified . depending on the volume or tonnage of the chemical used , the level and range of information required varies , as shown in table 1 .
notably , k - reach does not incorporate many of the animal welfare provisions contained in its european counterpart regulation , e.g. , the article 1 requirement to promote alternatives to animal testing , the article 13 requirement to generate new data wherever possible by means other than vertebrate animal tests , the article 25 requirement to avoid unnecessary testing , and the annex xi rules for waiving or otherwise adapting standard data requirements . according to the list of tests required by k - reach shown in table 1 , for chemical substances manufactured or imported in volumes of 1000 tons or more per year , the data package must include 46 test items ; this entails a huge amount of money and resources .
furthermore , to generate health hazard and ecotoxicology packages using conventional test guideline methods , the animal use would be astronomical . in the worst case scenario
, the number of animals required per substance would be nearly 6000 , just for the assessment of human health effects , at a cost of more than 1.5 billion korean won . the number of animals required and the costs for each test are listed in table 2 .
given that about 300 chemicals are newly introduced into korean market every year , the use of animals would be enormous without any drastic changes .
statistics published recently by the korean ministry of agriculture , food and rural affairs ( mafra ) reflects a 36.7% increase in experimental animal use between 2012 and 2015 ( 2012 , 1834000 ; 2013 , 1967000 ; 2014 , 2412000 ; 2015 , 2507000 ) .
this problem is not unique to korea . following the enactment of reach in the eu
, the european chemicals agency has estimated that 30000 substances will be registered , requiring upwards of 3.9 million animals to be used in tests costing 1.6 billion ( us$ 2.3 billion ) .
however , others have suggested that the european chemicals agency s estimates may represent a substantial underestimation of both chemical registrations and animal use , projecting on the order of 68000 substance registrations and use of 54 million animals , respectively .
thus , the european public has demanded swift action by authorities and industry to ensure effective sharing of existing data , uptake of validated alternatives to animal tests , and other scientifically supported replacement , reduction or refinement ( 3r ) best practices .
conventional animal experiments have been developed and used on the premise that the responses of animals in the laboratory to chemicals can provide information to predict those of humans in the real world .
however , this basis is somewhat incorrect with respect to species differences in genetic expression , absorption , distribution , metabolism and excretion , organ susceptibility , immune responses and resistance or tolerance to xenobiotics .
this point has been explicitly criticized by leist and hartung , who stated that the human is not a 70 kg rat . indeed , rats , one of the most widely exploited experimental animal species , have different metabolic capacity and immune responses to humans , which seriously undermines the predictability of the experimental data .
another important point which diminishes the utility of animal experiments is that extreme conditions are generally employed as the worst case scenario in view of chemical exposure levels ( extremely high doses ranging from 10-fold to 1000-fold higher than human exposure levels ) , exposure route ( forced eyelid eversion for ocular irritant administration and closure for persistent exposure , or injection into sutured oral pouches to simulate oral mucosal exposure ) , stress ( unrealistically stressful conditions like cold or hot conditions ) , or disease states ( no animal naturally exhibits an asthmalike syndrome that is similar to the disease in humans ) .
although these experimental approaches may save time ( using a high dose to curtail the time factor ) and cost , they frequently produce unrealistic and inaccurate dose - response data for the prediction of human responses . exemplifying this , endocrine disrupting chemicals generally exhibit unconventional non - linear , bell - shaped or u - shaped dose - response patterns that can not be properly evaluated in high - dose animal experiments .
the dose - limiting toxicity of drug candidates observed in animal tests mostly do not appear in human clinical trials and the poor predictability of animal experiments has been observed across diverse target tissues . due to the failure of clinical trials by the appearance of unscreened toxicity during preclinical trials , enormous amounts of money and time are wasted . in this context , the development of more human - relevant and advanced methods is necessary to replace or at least to supplement traditional animal tests .
in addition to the major scientific drawbacks described above , animal tests have been criticized for their inherent cruelty , for being excessively time - intensive and resource - intensive , restrictive in the number of substances or mixtures that can be tested , and of little value in understanding the mechanistic underpinnings of toxicity in the species of ultimate interest .
the last century has witnessed unprecedented scientific and technological advances in biology , represented by human whole genome sequencing , the birth of functional genomics , computational biology , and high - speed robot automation of cell - based ( in vitro ) screening systems .
these innovations are being incorporated into a wide range of health and molecular / cellular biological research sectors , providing renewed vigor and inspiration in these areas .
numerous novel and revolutionary biochemical and molecular tools have been developed through this innovation , including high throughput assays , quantitative real time - polymerase chain reaction , flow cytometry , high content assays , gene transfection , reporter gene assays and tissue engineering .
in fact , these new tools have helped to understand how toxicants disrupt the normal physiology of the human body at the cellular and molecular levels , which has contributed to the birth of molecular and mechanistic toxicology .
the resulting predictions regarding human safety and the risk of a chemical are potentially more relevant to people than animal tests . in line with this , the toxicology for the 21st century or
tox21 strategy has been embarked on with a grand vision to innovate virtually all routine toxicity testing to be conducted in human cells or cell lines . these non - animal based new testing methods enable safety evaluations of a much larger number of substances , in a more rapid , efficient and cost - effective way .
most importantly , these methods are likely to be more relevant to toxicity in humans , as well as capable of identifying the cellular mechanisms of toxicity using fewer or no animals .
safety and regulatory science is one of most conservative and slowest - moving sectors in taking up new and novel methods , since many countries with diverse cultural and socioeconomic backgrounds must comply with new regulation through so - called international and inter - regional harmonization
. moreover , there are various stakeholders with conflicting interests even within a single country .
this is understandable , since important policies like drinking water standards , sewage control , emission limits , inclusion on positive or negative lists , and authorizing the use of chemicals are based upon safety evaluations and risk assessments , which often cost enormous amounts of money to achieve compliance , with serious risks to public health or the environment in the absence of compliance .
prior to implementation , a newly developed method must be compared regarding its relevance and reliability to the original gold standard method or targeted toxicity endpoint in humans in a validation trial , which can take as long as 10 years from the initial research and development steps .
the results of validation studies are then subject to regulatory adoption processes , involving appraisal by experts with multi - disciplinary backgrounds , often with the participation of more than one regulatory body from multiple countries .
international regulatory bodies or collaborative organizations that can represent a large number of countries , like the organization for economic cooperation and development ( oecd ) , the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use and the international organization for standardization have participated in these review and appraisal processes to accommodate the opinions of member countries . of these
, the oecd is the most active in publishing and updating new test guidelines ( tgs ) , along with integrated approaches to testing and assessment and other guidance materials , which 35 member countries can follow as the standardized methods .
many in vitro or in vivo tests with animal replacement , reduction or refinement potential have been included as tgs recently , as shown in table 3 , indicating 3r concepts applied to tgs , which are classified according to toxic endpoints .
while non - animal tests have been actively developed and widely used in skin or eye irritation tests , the alternative tests are not yet established for acute toxicity or reprovductive toxicity .
oecd tgs on toxicity tests were first developed in early 1980s , and have been revised continuously by 3r concepts and with the progression of science . from the 1980s to the early 2000s , mainly in vivo methods using animals
were adopted as tgs , and the pace of development or the appearance of new methods was slow . however , since the late 2000s , the tgs have been annually revised with 3r concepts , and many new alternative animal tests have been adopted ( table 3 ) , and many old or obsolete tgs can now be abandoned .
human biology - based in vitro methods are typically more relevant and predictive , less time - consuming , can accommodate larger number of chemicals or mixtures than traditional animal experiments , and can address the mechanism of toxicity using the framework of an adverse outcome pathway ( aop ) . by systemically
categorizing the biological events leading to adverse effects into key events between two points , i.e. , a molecular initiating event and an adverse outcome , the aop describes an adverse outcome following exposure to a chemical by a series of key events and key event relationships that describe the causal relationships between the key events .
aops are instrumental for establishing the toxic mechanism of an adverse outcome and the utilization of safety test data at the in vitro , in vivo and human levels for risk assessments and regulatory applications . in the best case scenario
, the number of animals saved by employing multiple in vitro tests along with limited in vivo tests will be in the thousands , which will significantly contribute to animal ethics without compromising predictive capacity or risking human health or environmental protection .
ultimately and ideally , all animal experiments , including those with refined or reduced use of animals , are to be changed into non - animal based methods .
these methods can be largely categorized into in chemico , ex vivo and in vitro methods . approved oecd tgs fall into these categories , as shown in table 4 .
since these methods largely employ test - tube or multi - well plate formats , the throughput is much higher than traditional animal experiments .
for example , the 3d reconstructed human cornea - like epithelium ( rhce ) method can evaluate the ocular irritancy of 10 test substances in one run , which takes three days from the delivery of the model .
this test is equivalent to 10 tests using the in vivo draize rabbit eye irritation test , costing the lives 30 of rabbits and taking at least 10 days from the delivery of the rabbits .
this test can be harnessed to assess multiple combinations of test conditions like different mixture ratios , diverse exposure scenarios and the addition of metabolic capacity using a feasible amount of time and money . moreover , through targeting a single molecular event on the aop framework , the conclusions of alternative methods may be more direct and straightforward , which is critical to address the mechanism of toxicity and extrapolation into human responses .
for example , in the direct peptide reactivity assay , an in chemico skin sensitization test that addresses the haptenization response of a substance , positive results indicate that the chemical is reactive and can form protein adducts .
of course , there are limitations and shortcomings of non - animal methods as well .
first , most of the alternatives have not advanced into the level of risk assessment , since they only give a qualitative yes or no answer , namely hazard identification .
even though some methods give quantitative data , their utility for potency classification or risk assessment has not been fully validated .
in addition , since the alternatives address mostly a single key event in the series of events constituting a larger aop , they can not provide the full mechanistic information regarding the final outcome of the initial exposure . in this case
, a combination of multiple in vitro assays can be used as an integrated approach to testing and assessment ( iata ) .
although many validated and internationally recognized nonanimal methods and strategies are now available and ready for immediate use in regulatory frameworks such as k - reach , their uptake by korean authorities continues to be less than optimal .
whereas eu - reach data requirements and guidance have been or are being amended to incorporate all available oecd 3r tgs and related best practices , such is not yet the case with k - reach .
mandatory 3r requirement under k - reach and other legislation for the uptake of new methods .
in addition , there is a lack of inter - ministerial cooperation regarding the uptake of new methods .
for example , the korean centre for the validation of alternative methods has been established under the ministry of food and drug safety ( mfds ) , but does not currently include participation from other regulatory or research agencies in the development , validation or regulatory review of non - animal based methods , indicating the inter - ministerial inefficiency and passive attitudes towards new and more advanced methods . delayed implementation of 3r - based alternatives , due to these inefficiencies , would lead to the unnecessary sacrifice of laboratory animals , even though they are not better than new alternatives for the protection of human health and the environment .
there are also duplicate experiments for substances that have already been tested and assigned ghs classifications , including chemicals that are known corrosives , fatal if inhaled or probable carcinogens .
the scientific basis for duplicating well - established test results and regulatory classifications is incomprehensible , particularly where vertebrate animal testing would be involved , and most notably where such testing ( e.g. , of corrosives ) would result in the most extreme pain and suffering for the animals involved . to ensure that the korean regulatory framework for chemicals , and the general approach to the validation and adoption of 3r best practices , do not fall farther behind those of other developed nations , k - reach data requirements and
tgs must be immediately and frequently amended on regular basis to incorporate all relevant oecd 3r tgs and related best practices , including mandatory data sharing , chemical grouping and readacross to ensure maximum possible reduction of vertebrate animal use and no duplication of existing test results .
one overarching regulatory body shall be formed to include all korean ministries with a stake in alternative methods and safety assessment , including the mfds , the ministry of environment ( moe ) , and the mafra to facilitate inter - ministerial cooperation toward the development of a comprehensive government strategy and funding framework for the reduction and replacement of animals used for toxicity testing and life sciences research .
the implementation of k - reach will contribute to the safe use of chemicals by identifying human health effects and ecotoxicity before their introduction to human society and the environment .
however , a huge number of laboratory animals are required to comply with the current korean guidelines , which is sometimes unnecessary and can be replaced with alternative methods .
therefore , in order for k - reach to be more practically implemented , 3r concepts and alternative to animal tests ( aats ) should be actively accepted , which has been advanced in the developed countries .
using aats for safety assessment is scientific , highly reliable and predictable and may be more human - relevant without inflicting pain and death in laboratory animals .
moreover , the application of aats may be more economical and effective than animal tests .
the implementation of aats is also a concern in other chemical sectors such as the food , cosmetics , drug and agrochemical industries . in this regard ,
the collaboration of the moe , the mfds , and mafra in korea is critical .
korean regulatory authorities should actively communicate and collaborate to follow global trends in safety assessments and rapidly developing alternative tests . | objectiveskorea s act on the registration and evaluation of chemicals ( k - reach ) was enacted for the protection of human health and the environment in 2015 . considering that about 2000 new substances are introduced annually across the globe , the extent of animal testing requirement could be overwhelming unless regulators and companies work proactively to institute and enforce global best practices to replace , reduce or refine animal use . in this review , the way to reduce the animal use for k - reach is discussed.methodsbackground of the enforcement of the k - reach and its details was reviewed along with the papers and regulatory documents regarding the limitation of animal experiments and its alternatives in order to discuss the regulatory adoption of alternative tests.resultsdepending on the tonnage of the chemical used , the data required ranges from acute and other short - term studies for a single exposure route to testing via multiple exposure routes and costly , longer - term studies such as a full two - generation reproducibility toxicity .
the european registration , evaluation , authorization and restriction of chemicals regulation provides for mandatory sharing of vertebrate test data to avoid unnecessary duplication of animal use and test costs , and obligation to revise data requirements and test guidelines as soon as possible after relevant , validated replacement , reduction or refinement ( 3r ) methods become available .
furthermore , the organization for economic cooperation and development actively accepts alternative animal tests and 3r to chemical toxicity tests.conclusionsalternative tests which are more ethical and efficient than animal experiments should be widely used to assess the toxicity of chemicals for k - reach registration .
the relevant regulatory agencies will have to make efforts to actively adopt and uptake new alternative tests and 3r to k - reach . |
ficoll - isolated mononuclear peripheral blood ( pb ) cells from healthy donors were stained with abs against the lin markers cd3 , cd19 and cd56 , as well as with hla - dr , cd14 , cd11c , cd141 , cd1c , cd303 and the indicated workshop abs ( table 1 ) .
pdcs and two distinct myeloid dcs ( cd1c dcs , cd141 dcs ) were defined by multicolour immunofluorescence according to the flow cytometry gate setting strategy shown in figure 1 .
all dc subsets are characterized by the absence of the lin markers and cd14 and the presence of hla - dr and cd11c .
mdc2 were additionally selected by their absence of cd1c . based on this multigating strategy ,
pdcs express the key marker cd303 ( bdca-2 ) , whereas the myeloid dc subsets express the key markers cd1c ( bdca-1 ) and cd141 ( bdca-3 ) .
as demonstrated in figure 1 , mdc1 and mdc2 express high or medium levels of cd11c , respectively , whereas cd11c is only weakly expressed by pdc .
for profiling of dc subsets with workshop abs , plots of cd1c , cd141 or cd303 versus the selected monoclonal antibody ( mab)-defined antigens were compared with the corresponding controls . in figure 2
the profiles demonstrate that cd370 ( clec9a / dngr-1 ) is almost exclusively expressed on cd141 dcs , whereas cd366 ( tim-3 ) and cd101 ( table 1 ) expression is restricted to both myeloid dc subsets , and cd371 ( clec12a , cll , micl ) is expressed on all three dc subsets .
il-13-ra2 and vimentin , as well as two ab - defined antigens of unknown targets , show heterogeneous reactivity profiles on either the two myeloid dc subsets ( 10 - 30/il-13-ra2/clone fmu - il-13ra2 - 7 and 10 - 69/clone cmrf-56 ) or on all three dc subsets ( 10 - 55/vimentin / clone sc5 and 10 - 25/clone w4a5 ) ( table 1 ) .
this suggests that all three dc populations contain additional subsets with probably unknown function . of the new clustered molecules at the hlda10 conference , cd365 ( tim-1 )
is not expressed on primary dcs and weak cd368 ( clec4d , dectin-3 ) expression on these cells is only verified by one of two submitted abs ( positive with clone 9b9 ( 10 - 78 ) , but not with clone fab2806p ( 10 - 21 ) ) .
cd367 ( clec4a , dcir ) and cd369 ( clec7a , dectin-1 ) are weakly expressed on all dc subsets shown by staining with two out of three cd367-reactive abs ( clones fab1748p ( 10 - 13 ) and 9e8 ( 10 - 72 ) ) . in contrast , anti - cd369 abs showed clone - dependent reactivity patterns to peripheral blood dcs .
clone ge2 was not reactive to all three analysed dc populations , whereas clone fab1859p ( 10 - 35 ) interacted weakly with cd141 dcs and clone 15e2 ( 10 - 79 ) weakly with pdcs , cd1c and cd141 dcs ; table 1 ) .
the reactivity profiles of all available workshop mabs to dc subsets are summarized in table 1 .
workshop mabs were additionally analysed for their recognition of mo - dcs , which were obtained after culture of peripheral blood mononuclear cells ( pbmcs ) for 7 days in the presence of 100 ng ml gm - csf and 20 ng ml il-4 .
after culture , cells were double - stained with anti - cd209 ( dc - sign)-apc and the indicated workshop abs and analysed by flow cytometry .
figure 3 shows that cultured dcs reside in the monocyte ' gate in the dual scatter plot , which was verified by their expression of cd209 . after gating
, cells were analysed by plotting cd209 versus workshop mab and compared with appropriate controls .
the complete set of new cd molecules clustered at the hlda10 conference ( cd365 - 371 ) is expressed on mo - dcs at varying intensity . however
, some abs such as 10 - 1 ( cd369 ) , 10 - 2 ( cd370 ) and 10 - 17 ( cd371 ) did not recognize the epitopes on mo - dcs , although other workshop mabs against these molecules showed a positive staining on these cells .
mab against cd273 and cd276 , as well as the ab - defined antigens 58b3 , f9 - 3c2 ( 10 - 39 ) , w4a5 ( 10 - 25 ) and w5c4 ( 10 - 32 ) show unique reactivity patterns ( figure 3 ) .
in particular , mab 10 - 61 ( cd273 ) , mab 10 - 69 ( cmrf-56 ) , mab 10 - 39 ( f9 - 3c2 ) and mab 10 - 25 ( w4a5 ) give rise to heterogeneous reactivity patterns , suggesting that cultured dcs do not consist of a homogeneous cell population .
the reactivity profiles on mo - dc of all available workshop mab are summarized in table 2 .
ficoll - isolated mononuclear peripheral blood ( pb ) cells from healthy donors were stained with abs against the lin markers cd3 , cd19 and cd56 , as well as with hla - dr , cd14 , cd11c , cd141 , cd1c , cd303 and the indicated workshop abs ( table 1 ) .
pdcs and two distinct myeloid dcs ( cd1c dcs , cd141 dcs ) were defined by multicolour immunofluorescence according to the flow cytometry gate setting strategy shown in figure 1 .
all dc subsets are characterized by the absence of the lin markers and cd14 and the presence of hla - dr and cd11c .
mdc2 were additionally selected by their absence of cd1c . based on this multigating strategy ,
pdcs express the key marker cd303 ( bdca-2 ) , whereas the myeloid dc subsets express the key markers cd1c ( bdca-1 ) and cd141 ( bdca-3 ) .
as demonstrated in figure 1 , mdc1 and mdc2 express high or medium levels of cd11c , respectively , whereas cd11c is only weakly expressed by pdc .
for profiling of dc subsets with workshop abs , plots of cd1c , cd141 or cd303 versus the selected monoclonal antibody ( mab)-defined antigens were compared with the corresponding controls . in figure 2
the profiles demonstrate that cd370 ( clec9a / dngr-1 ) is almost exclusively expressed on cd141 dcs , whereas cd366 ( tim-3 ) and cd101 ( table 1 ) expression is restricted to both myeloid dc subsets , and cd371 ( clec12a , cll , micl ) is expressed on all three dc subsets .
il-13-ra2 and vimentin , as well as two ab - defined antigens of unknown targets , show heterogeneous reactivity profiles on either the two myeloid dc subsets ( 10 - 30/il-13-ra2/clone fmu - il-13ra2 - 7 and 10 - 69/clone cmrf-56 ) or on all three dc subsets ( 10 - 55/vimentin / clone sc5 and 10 - 25/clone w4a5 ) ( table 1 ) .
this suggests that all three dc populations contain additional subsets with probably unknown function . of the new clustered molecules at the hlda10 conference , cd365 ( tim-1 )
is not expressed on primary dcs and weak cd368 ( clec4d , dectin-3 ) expression on these cells is only verified by one of two submitted abs ( positive with clone 9b9 ( 10 - 78 ) , but not with clone fab2806p ( 10 - 21 ) ) .
cd367 ( clec4a , dcir ) and cd369 ( clec7a , dectin-1 ) are weakly expressed on all dc subsets shown by staining with two out of three cd367-reactive abs ( clones fab1748p ( 10 - 13 ) and 9e8 ( 10 - 72 ) ) . in contrast , anti - cd369 abs showed clone - dependent reactivity patterns to peripheral blood dcs .
clone ge2 was not reactive to all three analysed dc populations , whereas clone fab1859p ( 10 - 35 ) interacted weakly with cd141 dcs and clone 15e2 ( 10 - 79 ) weakly with pdcs , cd1c and cd141 dcs ; table 1 ) .
the reactivity profiles of all available workshop mabs to dc subsets are summarized in table 1 .
workshop mabs were additionally analysed for their recognition of mo - dcs , which were obtained after culture of peripheral blood mononuclear cells ( pbmcs ) for 7 days in the presence of 100 ng ml gm - csf and 20 ng ml il-4 . after culture ,
cells were double - stained with anti - cd209 ( dc - sign)-apc and the indicated workshop abs and analysed by flow cytometry .
figure 3 shows that cultured dcs reside in the monocyte ' gate in the dual scatter plot , which was verified by their expression of cd209 .
after gating , cells were analysed by plotting cd209 versus workshop mab and compared with appropriate controls .
the complete set of new cd molecules clustered at the hlda10 conference ( cd365 - 371 ) is expressed on mo - dcs at varying intensity .
however , some abs such as 10 - 1 ( cd369 ) , 10 - 2 ( cd370 ) and 10 - 17 ( cd371 ) did not recognize the epitopes on mo - dcs , although other workshop mabs against these molecules showed a positive staining on these cells .
mab against cd273 and cd276 , as well as the ab - defined antigens 58b3 , f9 - 3c2 ( 10 - 39 ) , w4a5 ( 10 - 25 ) and w5c4 ( 10 - 32 ) show unique reactivity patterns ( figure 3 ) .
in particular , mab 10 - 61 ( cd273 ) , mab 10 - 69 ( cmrf-56 ) , mab 10 - 39 ( f9 - 3c2 ) and mab 10 - 25 ( w4a5 ) give rise to heterogeneous reactivity patterns , suggesting that cultured dcs do not consist of a homogeneous cell population .
the reactivity profiles on mo - dc of all available workshop mab are summarized in table 2 .
we screened abs submitted from different providers to the hlda10 workshop in wollongong , australia , for their reactivity with primary human blood dc subsets as well as with in vitro generated mo - dcs .
the abs were either purified , delivered as ascites or directly conjugated with fitc ( fluorescein isothiocyanate ) or pe ( phycoerythrin ) and assigned to consecutive numbers .
two new abs cluster to single - span class-1 transmembrane proteins , namely cd365 ( tim-1 ) and cd366 ( tim-3 ) .
tim-3 has a critical role in regulating the activities of t cells , macrophages , monocytes , dcs , mast cells , natural killer cells and endothelial cells . whether these molecules are also expressed on human blood dc subsets was not yet investigated . here
we could show that cd365 is also weakly expressed on primary blood pdcs , cd1c and cd141 dcs , as well as on mo - dcs .
in contrast , cd366 is highly expressed on cd1c and cd141 dcs , as well as mo - dcs , but only near to background levels on pdcs .
these data are in contrast to a recent study showing no cd366 expression on mo - dcs from healthy volunteers or patients with different tumours , whereas tumour - associated cd11c dcs express high levels of cd366 .
this study did not discriminate between different subsets of tumour - associated cd11c dcs nor analysed peripheral blood dcs .
we could show that all four hlda10 abs generated against cd370 ( clec9a ) specifically recognize cd141 dcs , although abs 10 - 45 and 10 - 65 weakly reacted with pdcs , cd1c dcs and in vitro generated mo - dcs .
cd370 functions as an endocytic receptor specific for the uptake of degraded components from dead cells and is specifically expressed by cd141 dcs . the reasons for the occasionally decreased performance of workshop abs can be several : first
second , the quality of abs varied considerably , some were shipped as purified , some as culture supernatant , some as ascites fluid and others as fitc or pe conjugates .
third , many abs could be visualized only by indirect staining in combination with multicolour immunofluorescence analysis . as a result ,
the weaker binding of abs 10 - 45 and 10 - 65 compared with 10 - 02 and 10 - 09 can be explained by these reasons .
this is of particular evidence with regard to abs 10 - 02 and 10 - 65 , as these reagents are derived from the same clone . and
finally , different epitopes of the same molecule could be differentially expressed on the cell surface of primary and cultured dcs , resulting in a differential recognition of molecules by abs with the same molecule specificity .
cd369 ( clec7a / dectin-1 ) is an another pattern recognition receptor specific for -glucans , components of pathogenic bacteria and fungi and is expressed on dcs .
we could validate that the submitted cd370-reactive abs bind to primary blood cd1c dcs and cd141 dcs , as well as to in vitro generated mo - dcs , but not to pdcs .
in addition , all submitted abs against the c - type lectins including cd367 ( clec4a / dcir ) , cd368 ( clec4d ) and cd371 ( clec12a / micl ) bound to all dc subsets analysed .
cd367 expression is decreased upon maturation , but because of limited amount of abs provided , we were not able to confirm these findings
. finally , we identified three abs recognizing targets that are heterogeneously expressed on all primary blood dc subsets including vimentin , ab 10 - 69 and ab w4a5 .
although the latter two targets are not yet identified , they appear to be of particular interest because they are able to subdivide the subsets into either further sub - populations or into defined activation stages . in conclusion , using abs submitted to the hlda10 workshop , we could not only confirm the expression patterns of known dc targets but also expand the knowledge of molecules expressed on dcs by either demonstrating new reactivity patterns of known targets or presenting heterogeneous expression profiles of known and unknown targets on dc subsets .
peripheral blood was collected from two healthy donors according to the guidelines of the local ethics committee .
when fluorochrome - conjugated workshop abs were available , cells were incubated with a mixture of all ab conjugates for 30 min at 4 c .
conjugates consisted of the lin markers cd3-pacific blue ( sk7 ) , cd19-pacific blue ( hlb19 ) and cd56-pacific blue ( mem-188 ) , as well as of anti - hla - dr - brilliant violet 510 ( l243 ) , cd14-fitc ( m5e2 ) , cd11c - pe - cy7 ( bu15 ) , cd141-apc ( m80 ) , cd1c - apc - cy7 ( l161 ) and cd303-percp - cy5.5 ( 201 a ) ( purchased from biolegend inc . , san diego , ca , usa ) , and of pe - conjugated workshop abs . after washing with facs buffer ( phosphate - buffered saline , 0.1% nan3 , 1% bovine serum albumin ) ,
cells were additionally stained for 10 min with 3 m dapi ( 4',6-diamidino-2-phenylindole)at room temperature ( sigma - aldrich , saint louis , mo , usa ) for dead cell exclusion and acquired on a facscanto ii analyser ( heidelberg , germany ) .
stored data were analysed using the flowjo software programme ( flowjo , llc , ashland , or , usa ) .
the gating and plotting strategy to define pdc , cd1c dcs and cd141 dcs is shown in figure 1 . in the case where only non - conjugated
after washing , cells were stained with pe - conjugated goat anti - mouse antisera ( biozol diagnostica gmbh , eching , germany ) . in the next step
non - reacting ' mouse ab ( biozol diagnostica gmbh ) to block free binding sites of the secondary - step reagent . in the final step
, cells were incubated with the commercially available ab conjugates . for generation of mo - dcs
cells were seeded ( 1 10/10 ml ) into 75 cm cell culture flasks ( corning , cambridge , ma , usa ) in serum - free x - vivo 20 medium ( cambrex bio science , verviers , belgium ) .
after 2 h of incubation at 37 c/5% co2 , non - adherent cells were removed .
the monocytes were cultured in 10 ml rp10 medium ( rpmi-1640 with glutamax - i ) , supplemented with 10% inactivated foetal calf serum and antibiotics ( invitrogen , karlsruhe , germany ) .
gm - csf ( 100 ng ml ; leukine liquid sargramostim ; sanofi , bridgewater , nj , usa ) and il-4 ( 20 ng ml ; r&d systems , wiesbaden , germany ) were added every second day for 7 days .
after washing two times with facs buffer , cells were stained with allophycocyanin - conjugated anti - cd209 ab ( dcn46 ; bd biosciences , heidelberg , germany ) and the indicated workshop abs using the protocol described above .
peripheral blood was collected from two healthy donors according to the guidelines of the local ethics committee .
when fluorochrome - conjugated workshop abs were available , cells were incubated with a mixture of all ab conjugates for 30 min at 4 c .
conjugates consisted of the lin markers cd3-pacific blue ( sk7 ) , cd19-pacific blue ( hlb19 ) and cd56-pacific blue ( mem-188 ) , as well as of anti - hla - dr - brilliant violet 510 ( l243 ) , cd14-fitc ( m5e2 ) , cd11c - pe - cy7 ( bu15 ) , cd141-apc ( m80 ) , cd1c - apc - cy7 ( l161 ) and cd303-percp - cy5.5 ( 201 a ) ( purchased from biolegend inc . , san diego , ca , usa ) , and of pe - conjugated workshop abs . after washing with facs buffer ( phosphate - buffered saline , 0.1% nan3 , 1% bovine serum albumin ) ,
cells were additionally stained for 10 min with 3 m dapi ( 4',6-diamidino-2-phenylindole)at room temperature ( sigma - aldrich , saint louis , mo , usa ) for dead cell exclusion and acquired on a facscanto ii analyser ( heidelberg , germany ) .
stored data were analysed using the flowjo software programme ( flowjo , llc , ashland , or , usa ) .
the gating and plotting strategy to define pdc , cd1c dcs and cd141 dcs is shown in figure 1 . in the case where only non - conjugated
after washing , cells were stained with pe - conjugated goat anti - mouse antisera ( biozol diagnostica gmbh , eching , germany ) . in the next step
non - reacting ' mouse ab ( biozol diagnostica gmbh ) to block free binding sites of the secondary - step reagent . in the final step
for generation of mo - dcs , monocytes were purified by plastic adherence of pbmcs . for this purpose ,
cells were seeded ( 1 10/10 ml ) into 75 cm cell culture flasks ( corning , cambridge , ma , usa ) in serum - free x - vivo 20 medium ( cambrex bio science , verviers , belgium ) .
after 2 h of incubation at 37 c/5% co2 , non - adherent cells were removed .
the monocytes were cultured in 10 ml rp10 medium ( rpmi-1640 with glutamax - i ) , supplemented with 10% inactivated foetal calf serum and antibiotics ( invitrogen , karlsruhe , germany ) .
gm - csf ( 100 ng ml ; leukine liquid sargramostim ; sanofi , bridgewater , nj , usa ) and il-4 ( 20 ng ml ; r&d systems , wiesbaden , germany ) were added every second day for 7 days .
after washing two times with facs buffer , cells were stained with allophycocyanin - conjugated anti - cd209 ab ( dcn46 ; bd biosciences , heidelberg , germany ) and the indicated workshop abs using the protocol described above . | dendritic cells ( dcs ) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes . mainly three phenotypically and functionally distinct dc subsets
are described in the human peripheral blood ( pb ) : plasmacytoid dcs ( pdcs ) , which express the key marker cd303 ( bdca-2 ) , and two myeloid dc subsets ( cd1c+ dc ( mdc1 ) and cd141 + dc ( mdc2 ) ) , which express the key markers cd1c ( bdca-1 ) and cd141 ( bdca-3 ) , respectively .
in addition to these primary cell subsets , dcs can also be generated in vitro from either cd34 + stem / progenitor cells in the presence of flt3 ( fms - related tyrosine kinase 3 ) ligand or from cd14 + monocytes ( monocyte - derived dcs ( mo - dcs ) ) in the presence of granulocyte macrophage colony - stimulating factor+interleukin-4 ( gm - csf+il-4 ) .
here we compare the reactivity patterns of hlda10 antibodies ( monoclonal antibody ( mab ) ) with pdcs , cd1c+ dcs and cd141 + dcs , as well as with cd14 + -derived mo - dcs cultured for 7 days in the presence of 100 ng / ml gm - csf plus 20 ng / ml il-4 . a detailed profiling of these dc subsets based on immunophenotyping and multicolour flow cytometry analysis is presented . using the panel of hlda10 workshop mab , we could verify known targets selectively expressed on discrete dc subsets including cd370 as a selective marker for cd141 + dcs and cd366 as a marker for both myeloid subsets .
in addition , vimentin and other markers are heterogeneously expressed on all three subsets , suggesting the existence of so far not identified dc subsets . |
the size class of meiofauna is generally defined as the portion of the community passing through a 1-mm sieve and being retained on a 32-m sieve .
this community comprises protists and metazoan animals that remain small even as adults ( permanent meiofauna ) and animals that temporarily belong to this size class during their larval / juvenile development ( temporary meiofauna ) . as part of the sediment infauna
, meiobenthos has been studied extensively worldwide from many different habitats , but less attention has been paid to the hard - substrate epibenthic or epizooic and epiphytal meiobenthos ( giere , 2009 ) . at cold seeps , a variety of geologically diverse
, reducing habitats can be distinguished by the presence of microbial mats or macro / megafaunal communities ( see sibuet and olu , 1998 ; levin , 2005 ) .
although some animals , such as thyrasid bivalves or siboglinids , inhabit the sediment with only the anterior part of their tubes extending above the sediment surface , siboglinid vestimentiferans , bathymodiolin and vesicomyid bivalves , and sponges can build large physical structures above the sediment surface to create habitat as foundation species for an associated macro- and meiofaunal community .
in general , foundation species influence the abundance , composition , and structure of the associated community ( hacker and gaines , 1997 ) and can provide food resources , living space , favorable settlement conditions , refuge from predators , and/or refuge from environmental stress ( see bruno and bertness , 2001 ) .
the gulf of mexico ( gom ) was the site of the first discoveries of cold seeps in the 1980s ( paull et al . , 1984 ; kennicutt et al . , 1985 ) , and
most studies have been completed at seeps from the upper slope , located shallower than 1000 m , but more recently some have included deeper sites from the lower slope ( see cordes et al .
tubeworm bushes , composed mainly of mixed vestimentiferan populations of lamellibrachia lymesi and seepiophila jonesi , were studied from the upper louisiana slope ( bergquist et al . , 2003 ; cordes et al . , 2005 ) .
the tube surface area , taken as a measure of habitat size , increased the overall surface between 2.6- and 26-fold over the uncolonized seafloor ( bergquist et al . , 2003 ) . in deeper waters of the lower slope , studied vestimentiferan aggregations were composed primarily of escarpia laminata ( brooks et al .
there are a number of foundation species of mussels on the lower slope , and beds may consist of single species such as bathymodiolus brooksi ( at atwater valley ) and bathymodiolus childressi ( mississippi canyon ) , or mixed populations of b. brooksi and b. childressi ( alaminos canyon ) or b. brooksi and bathymodiolus heckerae ( florida escarpment ) ( cordes et al . , 2007 ) .
meiobenthic community studies at cold seeps are scarce and restricted mainly to assessments of abundance , biomass , and composition of higher taxa .
they cover a wide geographical and depth range , from shallow - water sands at 10 m down to deep - sea muds at 5000 m. they include various types of hydrocarbon gas and oil seep ( montagna and spies , 1985 ; palmer et al . , 1988 ; shirayama and ohta , 1990 ; olu et al . , 1997 ; robinson et al . , 2004
van gaever et al . , 2006 ; sergeeva and gulin , 2007 ; sommer et al . , 2007 ) ,
gas , oil , and asphalt seeps ( montagna et al . , 1987 ) , gas hydrates ( sommer et al . , 2007 ) , and brine seeps ( powell et al . , 1983 , 1986 ) ,
but exclusively describe the infaunal meiobenthos from sediments covered by bacterial mats , vesicomyid clams , siboglinid frenulates , and sclerolinum in the periphery of mussel beds .
furthermore , some sediments with discharge of methane but devoid of any visible microbial mat or animals have also been studied . in the gulf of mexico , seep meiofauna studies were conducted for the shallow brine seep sand communities at east flower garden ( powell et al . , 1983 , 1986 ; jensen , 1986 ) and the hydrocarbon seep bacterial mat communities at alaminos canyon ( 2200 m ) , green canyon ( about 700 m ) , and atwater valley ( about 2000 m ) ( robinson et al . , 2004 ) .
epifaunal foraminiferan communities associated with tubeworm bushes on the upper slope were also examined in detail ( sen gupta et al . , 2007 ) , but no study on the associated metazoan meiobenthos has been carried out so far .
this study examines the abundance and higher taxonomic composition of epizooic , permanent , metazoan meiobenthos associated with aggregations of tubeworms and mussels from three different locations : green canyon ( gc ) , atwater valley ( av ) , and alaminos canyon ( ac ) in the gulf of mexico .
in addition , non - seep sediment cores were taken in the vicinity of such aggregations at gc .
the following questions were addressed : ( 1 ) do abundance and higher taxonomic composition differ between geographical regions ? ( 2 )
do abundance and higher taxonomic composition differ between mussel and tubeworm aggregations ? ( 3 ) is the seep epizooic metazoan meiobenthic community similar to seep infauna or non - seep sediments ? ( 4 ) are there similarities in abundance and higher taxonomic composition of seep and hydrothermal vent communities associated with mussels and tubeworms ?
the study was conducted at the three hydrocarbon seep locations : green canyon 852 ( gc , depth 1400 m ) , alaminos canyon 818 ( ac , depth 2800 m ) , and atwater valley 340 ( av , depth 2200 m ) of the lower continental slope of the gulf of mexico ( this issue ) . during two cruises in 2006 and 2007 ,
a total of 13 samples were taken with the submersible dsv alvin ( 2006 ) and rov jason ( 2007 ) .
five samples of each foundation group were collected at two different seep habitats : mussels , m - gc1 , m - gc2 , m - gc3 , m - av1 , m - ac1 and tubeworms , t - gc1 , t - gc2 , t - gc3 , t - av1 , t - av2 .
three samples of non - seep sediments were taken as controls ( s - gc1 , s - gc2 , s - gc3 ) in close vicinity ( < 3-m distance ) to seep megafauna communities ( table 1 ) .
epifauna collections were carried out with the quantitative sampling devices mussel pot ( a hydraulically actuated sampling pot lined with a net , 531 cm diameter of sample area ; for further detail of the collection device see cordes et al . , 2010 ) for mussel aggregations and bushmaster jr .
( a hydraulically actuated , custom - built sampling device lined with a net and having a 2800-cm diameter of sample area ; for further details , see urcuyo et al . , 2003 ;
infauna of non - seep sediment were collected with push cores ( 6.3 cm diameter , 30 cm length ) .
samples were separately put into isolated , previously cleaned plastic boxes on the basket of dsv alvin or rov jason , transported to the surface , and recovered on deck of the rv atlantis in 2006 or noaa ship ron brown in 2007 . on board , the macro- and megafauna of bushmaster and mussel pot samples were carefully rinsed with cold 32-m - filtered seawater before we removed them from the samples in order to avoid loss of smaller fauna . mussels and tubeworms of each collection were identified and counted ( table 1 ) .
the samples were sieved through a 1-mm sieve to separate macro- from meiofauna . before sieving the samples through a 32-m sieve , we measured the volume of sediment of the entire sample < 1 mm .
the larger size fractions were retained for complementary studies by collaborators ( see cordes et al . , 2010 ) .
in order to estimate the sediment depth distribution of meiobenthos in the push corer samples , we checked the fraction deeper than 5 cm carefully on board of the ship .
because one sample lacked any specimens , and two samples contained only a single nematode , we took the upper 5 cm of these samples , and fixed them in 4% buffered formalin without sieving .
the push core sample s - gc1 was split into three parts along the entire length , and one part ( 52 ml ) was used for the present study .
the other two samples , s - gc2 and s - gc3 , were split into half , and these parts ( 78 ml ) were used for the present analyses . to extract meiofauna from the sediment , we used a density centrifugation technique with a medium consisting of a silicapolymer ( levasil ) mixed with kaolin ( mcintyre and warwick , 1984 ; veit - koehler et al . , 2008 ) .
except for sample t - av1 , all other samples were totally processed and the entire meiofauna community was counted and identified to higher taxon level .
sample t - av1 was extremely large ( 7.5 l sediment including meiofauna after sieving through a 1-mm net ) ; therefore , we carefully mixed the entire sample in a bucket , let it settle , randomly took a subsample of 217 ml , and estimated the total abundance from this subsample .
we noticed the presence of crustacean nauplii but did not include them in further analyses because they could not be assigned to a specific higher crustacean taxon .
we also recorded the protist meiobenthos , but did not include them in this study of permanent metazoan meiobenthos .
total abundance of meiobenthos was standardized to 10-cm sample area and additionally to 10-cm surface area of mussel shells and tubeworm tubes .
the surfaces of mussels and tubeworms were estimated for the main foundation species b. brooksi , b. childressi , b. heckerae , e. laminata , and lamellibrachia spp . by measurements of lengths and widths for each individual in the collection ( see cordes et al . , 2010 , for methods ) . to test for significant differences in abundances among habitat types in the green canyon samples , data were square - root transformed and bootstrapping was used , as this is a well - proven method when working with a relatively low number of samples and high variances ( 10,000 resamplings each , t - test , 2-sided test , routine ftboot from the package computer intensive statistics ( nemeschkal , 1999 ) .
results were classically bonferroni - corrected ( p=/n ; =0.05 ) . to evaluate similarity and dissimilarity among all samples , a bray
abundance data from 10-cm sample area were square - root transformed , but were not standardized , to enable us to better recognize differences caused by total abundances , and similarity percentage ( simper ) analysis , analysis of similarity ( anosim ) , and multi - dimensional scaling ( mds ) ordination were performed using primer v5 ( clarke and gorley , 2001 ) .
the total abundance of the permanent metazoan meiobenthos associated with mussel and tubeworm aggregations of most samples from three different locations at the northern gulf of mexico ranged from 1 to 8 1 individuals per 10-cm sample area .
however , one tubeworm aggregate sample ( t - av1 ) from atwater valley ( av ) revealed a total abundance between one and two orders of magnitude higher ( 447 individuals 10 cm ) than the nine other seep samples .
non - seep sediment control samples showed abundance values from 870 to 1523 individuals 10-cm sample area ( table 2 ) .
green canyon ( gc ) was the only site where the number of samples was sufficient to compare statistically the abundances among mussel- and tubeworm - associated communities , and among the seep communities and adjacent non - seep sediments .
we found no significant difference between mussel and tubeworm meiobenthos abundance ( p=0.190 ) , but significantly lower abundances at both seep communities than in non - seep sediments ( both : p=0.003 ) .
the mussel beds at av , ac , and one sample from gc ( m - gc3 ) were built exclusively by b. brooksi .
in addition , b. childressi co - occurred in two gc samples , contributing with 50% and 63.2% , respectively , to the total mussel abundance . also , the tubeworm aggregations of all collections were mixed populations of e. laminata and one or two species of lamellibrachia ( table 1 ) .
as foundation species forming biogenic habitat , tubeworms and mussels considerably increase the surface area and thus the potential living space for meiobenthos . by estimating the actual surface of the foundation species , we found an increase of surface in both types of aggregations between 1.78- and 6.03-fold .
the ratio of sample area to the surface area of tubes / shells was similar between the two biogenic habitat types , but was more variable in tubeworm bushes ( 1.786.03 ) than in mussel beds ( 3.075.46 ) ( table 1 ) . by assuming that the surface of foundation species was the actual living space of associated meiobenthos , we standardized the total abundance of this community to the surface area and calculated even lower densities , between only 1 and 3 individual per 10 cm .
again , one tubeworm sample ( t - av1 ) contained much greater densities of meiobenthos ( 20 individuals 10 cm ) ( table 2 ) .
t - tests on abundance per surface area of gc samples revealed similar results as calculations per sample area , with similarly low abundances found in the seep habitat types ( mussel and tubeworm : p=0.150 ; seep and non - seep both : p=0.003 ) .
the seep metazoan meiobenthic communities were composed of the higher taxa nematoda , copepoda , ostracoda , and halacarida .
in addition , nauplii larvae were found in seven out of ten samples with variable abundances but were excluded from analyses because it was impossible to assign them to a specific crustacean taxon .
the protist phylum foraminifera was also represented in all seep samples . in all five tubeworm samples from the three different locations ,
the most prominent taxa were the nematodes , with relative abundances between 57% and 90% , followed by the copepods ( 1043% ) .
ostracods and halacarids were relatively rare , often found with relative abundances below 1% and below 0.5% , respectively ( fig .
the relative distribution of higher taxa was more variable in mussel bed samples . in three samples ( m - gc3 , m - av1 , m - ac1 ) , nematodes dominated ( 6682% ) , followed by copepods ( 1730% ) , and in two samples ( m - gc1 , m - gc2 ) copepods were most abundant ( 82% and 99% ) .
ostracods were present in 4 out of 5 samples , and halacarids in 3 out of 5 samples . in two of these more diverse communities , ostracods and halacarids
the non - seep control sediments collected in close vicinity to mussel and tubeworm aggregations at gc additionally harbored the taxon kinorhyncha .
the community was composed primarily of nematodes ( 8081% ) , followed by copepods ( 1619% ) , ostracods , halacarids , and kinorhynchs ( all<1% ) .
simper and anosim analyses did not demonstrate significant differences between mussel bed and tubeworm aggregation meiobenthic communities at the taxonomic level examined .
there were also no significant differences among sites , despite the differences in depth ( gc 1400 m , av 2200 m , ac 2800 m ) ( table 3 ) .
however , strong differences were detected between non - seep sediment communities and tubeworm- and mussel - associated communities ( > 74% bray
curtis dissimilarity ) , and these differences were significant in the anosim ( r=0.64 ; p=0.040 for tubeworm / sediment ; r=0.81 ; p=0.020 for mussel / sediment ) .
multi - dimensional scaling ( mds ) ordination revealed that metazoan meiobenthos from seep habitats and from adjacent non - seep sediments formed distinct groups , with the exception of sample t - av1 , which exhibited relatively high similarity to non - seep communities ( fig .
the epizooic metazoan meiobenthic communities associated with tubeworm bushes and mussel beds at cold seeps in the gulf of mexico ( gom ) can be characterized as a community composed of a limited number of higher taxa , including nematoda , copepoda , ostracoda , and halacarida , occurring in remarkably low abundances .
these seep communities are similar to epizooic meiobenthic vent communities associated with bathymodiolin mussels or vestimentiferan tubeworms .
however , communities associated with biogenic habitats differ from the infaunal communities studied from sands of shallow - water seeps and clays of deep - water seeps , which show much higher abundances compared to the epizooic meiobenthos from our studied sites .
tubeworm aggregations and mussel beds are colonized not only by meiobenthos but also by a diverse and abundant macrobenthic community at the gom cold seeps . in these same samples ,
mussel - associated macrofauna were present in densities between 235.5 and 1196.3 individuals per m ( 0.2 and 1.2 individuals per 10 cm ) and tubeworm - associated macrofauna were between 35.9 and 127.9 individuals per m ( 0.04 and 1.3 individuals per 10 cm ) ( cordes et al . , 2010 ) .
in other samples from the upper slope , macrobenthic abundances calculated per sample area ranged from 209 to 9590 individuals per m ( 0.29 individuals per 10-cm ) ( bergquist et al . , 2003 ) , and abundances standardized to the tube surface vary from 4 to 233 individuals per m on the upper slope ( cordes et al . , 2005 ) , and 134607 individuals per m on the lower slope ( cordes et al . , 2007 )
abundances per mussel shell surface from the florida escarpment , a different site in atwater valley , and alaminos canyon were between 160 and 4458 individuals per m ( cordes et al . , 2007 ) .
it appears that the macro- and megafauna are relatively well represented in such aggregations , fueled by in situ primary production , whereas small meiobenthic animals are relatively scarce .
some shallow - water studies indicate that the interaction between macrofauna and meiofauna is negative for the smaller size class , because adult large animals are potentially predators and/or dislocate meiofauna by movement .
in addition , the juvenile macrofauna , temporarily in the meiofauna - size class while growing up , can act as predators or competitors ( bell , 1980 ; lafsson , 2003 ) . also , a recent study at seeps on the norwegian margin revealed a negative correlation between meio- and macrofaunal abundance , and predation pressure was speculated to be the underlying cause for this pattern ( van gaever et al . , 2009 ) .
however , whether the seep meiofauna community is regulated by such top - down or bottom - up processes remains to be tested .
overall , the abundances and higher taxonomic composition of meiobenthos associated with tubeworm and mussel habitats from cold seeps in this study are quite similar to those at hydrothermal vents ( table 4 ) .
the epizooic communities of both environments are low in abundance ( usually below 100 individuals per 10 cm ) and are mostly dominated by nematodes .
in addition , communities with equal nematode to copepod distribution ( east pacific rise , 950n region , tubeworm aggregations , gollner et al , 2007 ) , copepod - dominated communities ( this study ; juan de fuca ridge , paralvinella aggregations , tsurumi et al . , 2003 ; east pacific rise 11n region , mussel aggregations , zekely et al . , 2006 ; east pacific rise , 950n region , tubeworm aggregations , gollner et al . , 2007 ) , or foraminiferan - dominated communities ( east pacific rise , 950n region , tubeworm aggregations , gollner et al . , 2007 )
similar to varying higher taxa proportions in mussel aggregations at gc of this study , the tubeworm aggregation at the east pacific rise vent site riftia field also exhibited a high variability ( gollner et al . , 2007 ) .
this finding points to a patchy distribution , a common phenomenon , which other studies has been suggested to be related to the inhomogenous occurrence of food , predation , and/or displacement by larger animals ( see giere , 2009 ) . while the present study describes the epizooic meiobenthos from cold seeps , all other meiobenthic seep studies concern the infauna inhabiting seep sediments ( table 4 ) .
they range from very shallow sites down to 5000-m depth , come from different geographic regions and a variety of seep types , mostly hydrocarbon gas or gas / oil seeps but also gas / oil / asphalt seeps , gas hydrates , or brine seeps .
most samples were taken from sites covered by bacterial mats or colonized by siboglinid tubeworms , or were obtained from underneath clam beds , but sometimes from sites devoid of any microbial or megafaunal community .
in addition to different approaches in extraction techniques and size classes included in the meiofauna fraction , large variations also occur in studies in which only part of the meiobenthic community was analyzed .
some communities encompass the entire permanent ( metazoan and protist ) and temporary meiobenthos , and some include only parts .
overall , so far no trends in abundance according to depth , geographic regions , seep types , or habitat types are apparent .
epizooic metazoan meiobenthos from seeps ( 181 individuals per 10 cm ) and vents ( 1976 individuals per 10 cm ) , as well as vent infauna from sediments ( 11075 individuals per 10 cm ) , seem overall to be lower in abundance than infaunal meiobenthos from seeps ( 111292 individuals per 10 cm ) ( table 4 ) .
low abundances of seep infauna were detected only in anoxic sediments of the black sea and in some samples from a brine seep at east flower garden banks and at the norwegian margin ( powell et al .
, 1983 ; sergeeva and gulin , 2007 ; van gaever et al . , 2009 ) .
all other infaunal abundances are at least above 100 individuals per 10 cm and most exceed 1000 individuals per 10 cm ( table 4 ) .
the vast majority of epizooic and infaunal vent and seep meiobenthic samples are dominated by nematodes , usually followed by copepods .
other dominant taxa include gnathostomulids and plathelminths in highly sulfidic brine seep samples ( powell et al . , 1983 ) and rotifers in gas hydrate samples ( sommer et al . , 2007 )
although in several meiobenthic studies of seeps the nearby non - seep deep - sea samples were found to be lower in abundance than the seep sediment samples ( olu et al . , 1997 ; robinson et al . , 2004 ;
van gaever et al . , 2006 ) , our study could not confirm this trend .
in general , the abundance of meiobenthos in the deep sea has been found to decrease with depth owing to a decrease in pom flux in addition to sedimentary factors such as calcium carbonate content and sorting ( see soltwedel , 2000 ) .
ranges between 100 and 1000 individuals per 10 cm at shallower depths and between 10 and 100 individuals per 10 cm at deep sites are considered quite typical ( see giere , 2009 ) .
a very large data set from the gom deep - sea meiobenthos , carried out at between 200- and 3000 m depth , indicated a range between 600 and 9500 individuals per 10 cm ( baguley et al . ,
2006 ) . calculated from the correlation between abundance and depth , approximately 2500 individuals per 10 cm are expected in about 1500-m depth ( baguley et al . , 2006 ) .
this estimate is much higher than the actual abundances ( 8701523 individuals per 10 cm ) in our comparable non - seep sediments at a similar depth of 1450 m. the more puzzling result of this study , however , was the remarkably low abundances at seep sites .
the fact that meiobenthos associated with similar foundation species at vents is also low in abundance points to a commonality between seeps and vents , and is in sharp contrast to the high abundance of associated seep and vent macrobenthos . since in situ primary production
obviously fuels the large - sized community , it seems unlikely that meiobenthos is bottom - up controlled .
instead , the interactions with macrobenthos , such as high predation pressure and/or competition , are more likely to be underlying causes , but these hypotheses need to be tested .
also , detailed studies on the species richness and diversity patterns of these epizooic deep - sea communities are currently in progress and will help to elucidate the origin and evolution of seep meiobenthos . | the abundance and higher taxonomic composition of epizooic metazoan meiobenthic communities associated with mussel and tubeworm aggregations of hydrocarbon seeps at green canyon , atwater valley , and alaminos canyon in depths between 1400 and 2800 m were studied and compared to the infaunal community of non - seep sediments nearby .
epizooic meiofaunal abundances of associated meiobenthos living in tubeworm bushes and mussel beds at seeps were extremely low ( usually < 100 ind .
10 cm2 ) , similar to epizooic meiofauna at deep - sea hydrothermal vents , and the communities were composed primarily of nematodes , copepods , ostracods , and halacarids .
in contrast , epizooic meiobenthic abundance is lower than previous studies have reported for infauna from seep sediments .
interestingly , non - seep sediments contained higher abundances and higher taxonomic diversity than epizooic seep communities , although in situ primary production is restricted to seeps . |
age - related cataract is among the leading causes of severe visual impairment nowadays . with approximately 15 million cases performed annually ,
cataract extraction is one of the most frequent day - case procedures in medicine performed worldwide .
anticipated demographic changes ( population growth in developing countries and population ageing in both developing and developed countries ) will gradually increase the amount of cataract - related visual morbidity and double the cataract subject pool in 20-year time .
this steadily increasing need for surgical services capable of delivering good quality vision rehabilitation leads to a subsequent need for qualified , proficient ophthalmic surgeons . to accommodate this situation , effective and validated surgical training programs
are required to ensure that residents in training achieve greater levels of competence in cataract surgery .
wet laboratory training and simulators play an increasingly important role as part of an organized surgical curriculum [ 3 , 4 ] .
surgical competency assessment tools are also being developed and integrated in ophthalmic training and , in many residency programs , trainees ' point of view of the quality of training they receive is investigated [ 68 ] , as well as cataract surgery trainers ' attitudes and techniques . the learning curve for phacoemulsification cataract surgery
posterior capsule rupture with or without vitreous loss is considered to be the main undesirable intraoperative complication of phacoemulsification surgery and in most studies complication rates as well as visual outcomes are used as indicators of success . as a matter of fact ,
the incidence of posterior capsule ( pc ) tear among trainee surgeons varies from 5.8% to 15% [ 1012 ] and for pc tear with vitreous loss between 2.8% and 10% [ 1316 ] .
phacoemulsification is often taught in stages , where a trainee performs a single part of the procedure several times in succession under the direct supervision of an experienced surgeon .
when each step is mastered , the trainee then proceeds by learning the next step of the procedure .
so far , a number of teaching techniques have been described [ 12 , 17 ] ; however , to the best of our knowledge , there are no published studies in the literature directly comparing their efficacy on resident surgical outcomes .
the aim of this study is to evaluate and compare the efficacy of two different training methods in resident - performed phacoemulsification surgery and assess their impact on residents ' surgical training .
five - hundred - and - two eyes of 467 patients , who underwent resident - performed cataract surgery by means of phacoemulsification at a tertiary care centre from 2008 to 2013 , were included in a retrospective comparative study .
the study was conducted in accordance with the tenets of the declaration of helsinki and was approved by the local institutional review board . a retrospective analysis of all phacoemulsification cataract surgeries performed by ophthalmology residents from march 2008 to february 2013 in the 2nd department of ophthalmology , aristotle university of thessaloniki in papageorgiou general hospital , was conducted .
data were collected by reviewing patients ' electronic medical records . written informed consent to undergo cataract surgery was obtained from all patients and residents ' participation in the operation was clearly disclosed .
operations performed by residents with previous surgical experience in other hospitals were excluded from this study .
all surgical procedures were performed by 8 consecutive residents who did not have any previous experience as primary surgeon on intraocular procedures and completed at least 40 phacoemulsification surgeries .
patients with cataract causing visual disturbance and no history of significant risk factors were eligible for resident training .
additional exclusion criteria were ocular disease such as corneal opacity or irregularity , astigmatism greater than 2.5 d , severe dry eye syndrome , inadequate visualization of the fundus , iol power calculation less than 10.0 diopters ( d ) or more than 30.0 d , surgical complications on the fellow eye , and incomplete follow - up .
patients with a history of uveitis and current intraocular inflammation , uncontrollable glaucoma , proliferative diabetic retinopathy , or retinal detachment were also excluded .
phacoemulsification was performed in all cases under topical anesthesia with eye drops of proparacaine hydrochloride ophthalmic solution 0.5% ( alcaine , alcon laboratories , ca , usa ) using the infiniti vision system ( alcon inc . ,
all procedures were supervised by one high - volume attending surgeon ( it ) , who had the priority to take over in case of intraoperative complications or where the resident surgeon was unable to complete the procedure .
residents were categorized in two groups , group a and group b , according to the method of surgical training they received .
the allocation of residents to the training methods was conducted randomly and was not affected by unknown factors such as preferences of their supervisors .
all residents received equal traditional didactic and wet - lab training prior to operating on patient eyes .
group a residents were trained with a step - by - step method , performing a gradually increasing number of phacoemulsification steps during the course of their training . upon finishing each step successfully without any associated complications ( at least 10 times ) ,
steps were taught in the same sequence as they appear in phacoemulsification surgery ( corneal incisions ; capsulorhexis ; hydrodissection / hydrodelineation ; phacoemulsification ; irrigation / aspiration ; iol insertion / removal of viscoelastic device ) .
group b residents received a one - step method training program , during which they assisted in a minimum of five hundred ( 500 ) phacoemulsification operations and subsequently proceeded to perform the entire operation .
all residents were trained to use divide and conquer phacoemulsification technique , which was considered easier to learn and safer for the patient .
the first 40 consecutive cases that each resident performed independently as the primary surgeon were additionally studied in a separate analysis .
outcomes of surgeries performed by group a residents were compared with those of cases performed by their group b counterparts .
primary outcomes were the incidence of main complications , defined as posterior capsular ruptures and zonular dehiscence with vitreous loss , and other intraoperative complications ' rate .
statistical analysis was performed with medcalc statistical software ( version 9.3.0.0 , medcalc , mariakerke , belgium ) and spss ( v. 17.0 for windows , spss inc . , chicago , il ) .
normality was checked using the kolmogorov - smirnov test . due to the fact that data were not in all cases normally distributed
the association of not normally distributed sizes was assessed through rank correlation calculating spearman 's coefficient rho .
when parametric analysis was possible , the student t - test was used to compare the outcomes between two groups .
categorical variables were compared using the fisher exact probability test and chi - square test .
nonparametric mann - whitney u test was also used to examine associations between categorical variables and continuous or ordered outcomes .
eleven residents were trained during a 5-year time period between march 2008 and february 2013 .
data from 3 residents were excluded from statistical analysis , 2 due to previous surgical experience on phacoemulsification in other ophthalmology departments and 1 due to short surgical education ( 23 completed phacoemulsification surgeries ) not reaching the minimum surgical cases number ( 40 ) . in total , data from 8 residents were enrolled in the retrospective analysis . in total , the medical records of 502 phacoemulsification cases performed by 8 surgically naive residents were identified and reviewed .
all cases were allocated to two groups according to the method that was applied during the residents training in cataract surgery : group a included cases from 3 residents that were trained with the step - by - step method and group b included cases from 5 residents that were trained with the one - step method after assisting in at least 500 phacoemulsification cases performed by experienced instructors .
no preoperative statistically significant difference was noted in the main demographics and clinical characteristics data between two groups , which are summarized in table 1 . a statistically significant difference ( p = 0.0032 , fisher 's exact test )
was noted in the main complications rate between the two groups , yielding a mean of 17.3% in group a and 7.25% in group b , respectively .
other intraoperative complications , such as anterior capsule tear or extended capsulorhexis ( group a = 4.86% , group b = 4.42% ) , wound dehiscence ( group a = 1.62% , group b = 0.95% ) , and corneal lesions of any kind ( group a = 0.54% , group b = 0.32% ) , were not shown to differ statistically significantly between the two study groups ( p > 0.05 ) .
a detailed list of all the complications with the percentages for each study group and the respective p values is provided in table 2 . in order to minimize study bias originating from difference in the absolute number of procedures performed between the two study groups ,
a separate analysis was conducted including the first set of 40 consecutive cases performed by each resident , giving a total of 320 cases . among these cases , capsular tear rates differed also statistically significantly ( p = 0.0048 , fisher 's exact test ) between group a ( 21.67% ) and group b ( 8.5% ) , while a better surgical performance , yielding statistical significance in group a ( p = 0.017 , chi - square test ) and not in group b ( p = 0.07 , chi - square test ) , was indicated in both groups between the 20th and the 30th operation , where main complications rate was 7.5% in comparison to the chronologically 1st , 2nd , and 4th set of ten consecutive cases where this was assessed as 17.5% , 11.25% , and 17.5% , respectively .
complication rates for each study group and comparative data for the first 40 cases are shown in table 3 , whereas figure 1 illustrates the percentage of posterior capsular tears in each group dividing cases into sets of 10 as they were chronologically performed . a statistically significant negative correlation (
spearman 's r = 0.788 , p = 0.035 ) was noted between the number of complications that residents faced in their first 40 surgical cases and the total number of procedures that each one was allowed to complete . the correlation diagram is shown in figure 2 .
in a worldwide level , the demand for cataract surgery is expected to surge in the near future and this impending spike is multifactorial . aside from a sharp increase in the number of aging patients with cataracts , evolving surgical techniques and better surgical outcomes promise to boost demand among relatively young , active patients who desire early cataract surgery or refractive lens exchange . the key solution to accommodate
this situation is to enhance the efficiency and safety in current phacoemulsification training programs in order to generate better qualified cataract surgeons .
a variety of teaching methods and modalities have been described on phacoemulsification training [ 3 , 4 , 7 , 9 , 1215 ] .
traditional programs include studying basic literature on phacoemulsification , wet - lab practice , and starting phacoemulsification under supervision [ 9 , 14 ] .
another basic part of training in phacoemulsification is assisting in cases of experienced tutors . acting as a scrubbing assistant for more than several hundred cases offers candidates the ability to collect useful ideas pertaining to basic phacoemulsification processes , risk evaluation , and recognition of early signs of complications and their management .
most phacoemulsification trainers fear that moving directly to resident - performed surgery would lead to higher complication rates . as a matter of fact
, many phaco tutors tend to allow an assistant to perform only some simple steps of the procedure , such as irrigation - aspiration and insertion of an iol , following successful practice on porcine eyes . to the best of our knowledge
though , there are no published studies directly comparing the efficacy of step - by - step method to one - step method in terms of intraoperative complications rate .
therefore , in our study , we attempted to assess and compare the efficacy of the above mentioned different training methods in resident - performed phacoemulsification surgery .
our results show that residents trained with the one - step method outperformed group a residents ( step - by - step teaching program ) , as shown by lower percentages of main intraoperative complications , defined as posterior capsular ruptures and zonular dehiscence with vitreous loss .
a statistically significant difference was noted in these complications ' rate between the two groups , yielding a mean of 17.3% in step - by - step group and 7.25% in one - step group .
taking into account only the first forty cases per resident the incidence of main complications was increased to 21.67% and 8.5% in the above mentioned groups , respectively , and pcr rate was 17.5% and 6% , respectively .
in a recently published study on step - by - step phacoemulsification training program for ophthalmology residents found lower posterior capsular rates ( 10.8 4.2% ) in the first 30 cases performed by each trainee .
there is a great variety of studies assessing the incidence of intraoperative complications in resident phacoemulsification surgeries .
hashemi et al . demonstrated that , among 500 cases operated by residents , 51 ( 10.2% ) developed vitreous loss and 48 ( 9.6% ) developed posterior capsule rupture and vitreous loss .
they concluded that direct attending supervision and careful case selection for the level of cataract surgery residency were of utmost importance in avoiding sight - threatening complications .
, trying to determine whether virtual surgery simulator training improves actual resident cataract surgery performance , found that capsular tear rates for the nonsimulator and simulator groups were 8.8% and 10% , respectively , for the first 25 cases .
studying resident - performed phacoemulsification surgery , indicated a statistically significant difference in pcr rates between the first 40 cases per resident and the total number of operations , yielding a mean of 9.65% in the first 40 operations , data comparable to our results .
several other studies showed lower complications rates in residents training programs varying from 3% to 7% [ 2327 ] .
this difference may be attributed to the fact that residents included in these studies were not in all cases surgically naive and the complications rate was calculated for a larger amount of phacoemulsification cases performed by each resident , which could possibly lower the cumulative complications rate according to the learning curve .
the superior performance of residents trained with the one - step method may be attributed to a better perception of cataract surgery .
young ophthalmic surgeons are taught that an operation does not consist of a number of independent steps and they realize that every mistake , even the slightest , leads to increased difficulty and complication rates .
findings of the present study indicate that integration of the one - step method in ophthalmology training programs may lead to fewer complications and , therefore , to improved surgical outcomes .
obstacles such as the necessity of a long period of assistance in operations performed by experienced surgeons and trainers ' possible hesitation to allow a surgeon to undertake an operation from beginning to completion need to be overcome .
incorporation of the one - step method in resident training programs , as it has been demonstrated , may improve the quality of ophthalmology residency training and accommodate the increasing demand for skilled ophthalmic surgeons .
another major finding of the present study is that residents , during their first 40 operations , showed a better performance in terms of lower intraoperative complications rate in the 3rd set of 10 cases where main complications rate was 7.5% in comparison to 1st , 2nd , and 4th set where this was assessed as 17.5% , 11.25% , and 17.5% , respectively .
the above mentioned fluctuation in complications rate according to level of experience was noticed in both groups , independently of the training method used .
we believe this interesting remark may be attributed to the fact that most residents neglect several important rules once they feel confident enough to reduce operating times after their first 30 cases . the amount of phacoemulsification cases performed exclusively by an ophthalmology resident , or the completion rate ,
every supervisor should prevent some potentially dangerous or inappropriate movements and take over the case if serious intraoperative complications occurred .
supervisor 's attitude and alertness during residents ' operating attempts somewhat modify the incidence of complications and only this parameter could not reflect the real acquisition of skills in phacoemulsification .
a phacoemulsification trainer should strive to give equal support and opportunities to each resident during their training program [ 9 , 16 ] .
however , in a patient - based practice , most supervisors will try their best to avoid a high complication rate .
this common belief is in concordance with our finding that residents with lower complications rates in their first 40 cases were allowed to perform significantly more phacoemulsification surgeries during their residency programs
. limitations of our study include its retrospective design and the review of a single attending physician 's technique .
however , consistency in technique and instrumentation was important to analyze the impact of different teaching methods on residency cataract surgery training .
in addition , confounding data , such as ocular comorbidities , cataract characteristics of patients , and other possible risk factors , were not accounted for in this study .
furthermore , although surgical cases per resident as a mean did not differ statistically significantly between the two groups , there was a quite uneven distribution of performed phaco cases among trainees , ranging from 42 to 105 surgeries per resident .
based on the analysis , this was mainly attributed to the retrospective nature of the study and to the fact that residents with more complications had fewer surgical opportunities after the first 40 cases .
this finding could affect the overall complication rate and should be considered . in conclusion , to the best of our knowledge , this study is the first to compare directly the step - by - step to the one - step teaching methods in phacoemulsification training of residents .
the results of our study imply the fact that training in cataract surgery with the use of one - step method may enhance surgical performance , when measured by intraoperative complication rates , and , therefore , lead to a better quality of training for resident ophthalmologists , improving their learning curve .
further investigation analyzing the effect of various methods of resident surgical training on final corrected visual acuity is warranted in the future . | purpose . to evaluate and compare the efficacy of two different training methods in resident - performed phacoemulsification surgery
. methods .
502 eyes of 467 patients who underwent resident - performed phacoemulsification were included in the study by reviewing their medical records .
residents were allocated into two groups according to the method applied during their training in cataract surgery ; group a included residents that were trained with the step - by - step method and group b those trained with the one - step method .
primary outcome was the incidence of main complications , defined as posterior capsular ruptures and/or zonular dehiscence with vitreous loss .
results .
each resident performed a median of 63 phacoemulsification surgeries . a statistically significant difference ( p = 0.0032 )
was noted in the main complications rate between the two groups , yielding a mean of 17.3% in group a and 7.25% in group b. other intraoperative complications were not shown to differ statistically significantly between study groups ( p > 0.05 ) . among the first 40 surgeries of each resident , main complications rate differed also statistically significantly ( p = 0.0048 ) between group a ( 21.67% ) and group b ( 8.5% ) , while a better surgical performance - yielding statistical significance in group a ( p = 0.017 ) was indicated in both groups between the 20th and the 30th procedure .
conclusions .
training in cataract surgery using the one - step method may lead to an improvement in surgical competency , when measured by complications rates and , therefore , to significantly better quality of training for resident ophthalmologists . |
tourette syndrome ( ts ) , also known as multiple transient tic disorder or combined vocal and multiple motor tics , is a type of chronic neuropsychiatric developmental disorder that occurs in children and adolescents .
it is characterized by involuntary vocal tics and multiple motor tics of the head and body , particularly the face and extremities .
these symptoms may occur simultaneously or during different disease periods , showing a sudden and rapid onset , purposelessness , stereotypy , and arhythmicity .
the prevalence of ts has increased within recent years and is estimated to be 0.05%3% ; a large - scale epidemiological survey of 420,312 children and adolescents reported it to be 1.0% .
the ratio of the ts prevalence rate between males and females is 35:1 , and most patients develop the disease between the ages of 2 and 15 years , with a mean age of 7 years .
tic severity is closely related to age , with the most severe symptoms usually occurring at 812 years of age , causing a great impact on the children 's self - esteem , self - confidence , peer relationships , and family relationships .
about half of ts patients suffer social dysfunctions such as learning difficulties and interpersonal difficulties .
it is widely acknowledged that ts results from the interaction between genetic and environmental factors , as shown by pedigree and twin studies .
indeed , antipsychotic agents , both typical dopamine antagonists and atypical dopamine antagonists , have been proven to improve the symptoms of ts .
in addition , neuroimaging studies have found abnormalities in brain structures that are functionally related to the dopamine neurotransmission system .
therapeutic response to dopamine antagonists , as well as neuroimaging studies , supports a dopaminergic system abnormality in ts .
currently , the polymorphism of many dopaminergic system - related genes has been identified , which may be associated with ts .
the genes are the dopamine transporter 1 ( dat1 ) gene , the three d2-like dopamine receptor ( drd2 , drd3 , and drd4 ) genes and the monoamine oxidase - a gene .
still , a lack of replicated and consistent results was observed , a single nucleotide polymorphisms ( snps ) in these genes have a poor reproducibility .
moreover , existing studies have focused on drd2 , drd4 , and dat1 , while little is known about drd3 . in the past 10 years , we had focused on the study of exploring the association between the gene drd2 , drd4 , and dat between ts in a han chinese population .
there has not been any report about the association study of ts and drd3 gene in han chinese population . in this study
, we investigated the presence of the drd3 rs6280 polymorphism in ts patients , their unaffected parents , and normal controls , compared genotype and allele frequencies between ts patients and controls , and observed transmission disequilibrium between ts patients and their unaffected parents .
a total of 160 ts patients was recruited from the department of pediatrics , beijing anding hospital , capital medical university , china between december 2009 and december 2012 , including 101 nuclear pedigrees .
psychiatric diagnoses were made by a psychiatrist according to the diagnostic criteria of the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) .
the following inclusion criteria were used for the ts group : ( 1 ) meeting the diagnostic criteria of dsm - iv for ts ; ( 2 ) an age of 618 years ; ( 3 ) having no other central nervous system diseases or serious physical illness ; ( 4 ) a chinese han ; ( 5 ) having no tic disorder or other mental disorder patients in three generations of two pedigrees ; ( 6 ) being able to complete all diagnostic tests ; ( 7 ) providing informed consent .
the following inclusion criteria were used for the control group : ( 1 ) a healthy child or adolescent ; ( 2 ) an age of 618 years ; ( 3 ) having no central nervous system disease or severe physical illness , and no drug abuse or dependence ; ( 4 ) having no mental disorder , including attempted suicide , or family history of mental disorder ; ( 5 ) a chinese han individual unrelated to other controls or ts patients ; ( 6 ) a similar gender ratio to ts patients ; ( 7 ) being able to complete all diagnostic tests ; ( 8) providing informed consent .
the following inclusion criteria were used for the parents of ts patients : ( 1 ) biological parents of the ts patients ; ( 2 ) healthy individuals with no history of mental disorder and not meeting the dsm - iv diagnostic criteria ; ( 3 ) providing informed consent .
this experiment was approved by the ethics committee , beijing anding hospital , capital medical university , china .
the study was explained to all subjects prior to experimentation , and informed consent was obtained from all patients and their families .
the consent forms were signed by the guardians of subjects < 7 years old , and by both guardians and subjects 7 years old .
the work in this paper has been carried out in accordance with the code of ethics of the world medical association ( declaration of helsinki ) .
fasting peripheral blood samples ( 5 ml ) were collected in edta tubes , and genomic dna was extracted using the centrifugation column method with the whole blood genomic dna extraction kit ( promega ; madison , wi , usa ) according to the manufacturer 's instructions .
based on chinese han population genetics data from the database of snps ( http://www.ncbi.nlm.nih.gov/snp/ ; the us national center for biotechnology information ) , the international hapmap project ( http://www.hapmap.org/ ) , and previous studies , the drd3 snp rs6280 locus was selected for gene polymorphism analysis .
dopamine d3 receptor snp rs6280 genotyping was carried out using a taqman snp genotyping probe .
polymerase chain reaction ( pcr ) reactions ( 5 l ) contained 10 ng dna template , 2.5 l 2 taqman universal pcr master mix ( abi ) , and 0.25 l 20 snp assay ( applied biosystems , bedford , ma , usa ; including primers and fam / vic probe ) .
the reaction conditions were 95c for 10 min followed by 40 cycles of 92c for 15 s and 60c for 1 min .
pcr signals and fluorescence signals were read on an abi7900ht sequence detector ( 384-well plates ) , and then analyzed using the sds2.4 image analysis software ( abi prism , abi corp ) allelic discrimination program .
fam and vic fluorescence intensities of different alleles were analyzed to determine whether samples were homozygous wild - type , heterozygous or homozygous mutated .
spss 17.0 software ( ibm spss statistics for windows , ibm corp . , armonk , ny , usa ) was used for data analysis .
the fitness of the genotype and allele was tested using the hardy weinberg equilibrium ( hwe ) test , and polymorphism differences between the ts patient and control groups were analyzed using the chi - squared ( ) test .
the transmission disequilibrium test ( tdt ) of nuclear pedigrees was performed using plink1.07 software ( shaun purcell , usa ) , = 0.05 .
a total of 160 ts patients was recruited from the department of pediatrics , beijing anding hospital , capital medical university , china between december 2009 and december 2012 , including 101 nuclear pedigrees .
psychiatric diagnoses were made by a psychiatrist according to the diagnostic criteria of the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) .
the following inclusion criteria were used for the ts group : ( 1 ) meeting the diagnostic criteria of dsm - iv for ts ; ( 2 ) an age of 618 years ; ( 3 ) having no other central nervous system diseases or serious physical illness ; ( 4 ) a chinese han ; ( 5 ) having no tic disorder or other mental disorder patients in three generations of two pedigrees ; ( 6 ) being able to complete all diagnostic tests ; ( 7 ) providing informed consent .
the following inclusion criteria were used for the control group : ( 1 ) a healthy child or adolescent ; ( 2 ) an age of 618 years ; ( 3 ) having no central nervous system disease or severe physical illness , and no drug abuse or dependence ; ( 4 ) having no mental disorder , including attempted suicide , or family history of mental disorder ; ( 5 ) a chinese han individual unrelated to other controls or ts patients ; ( 6 ) a similar gender ratio to ts patients ; ( 7 ) being able to complete all diagnostic tests ; ( 8) providing informed consent .
the following inclusion criteria were used for the parents of ts patients : ( 1 ) biological parents of the ts patients ; ( 2 ) healthy individuals with no history of mental disorder and not meeting the dsm - iv diagnostic criteria ; ( 3 ) providing informed consent .
this experiment was approved by the ethics committee , beijing anding hospital , capital medical university , china .
the study was explained to all subjects prior to experimentation , and informed consent was obtained from all patients and their families .
the consent forms were signed by the guardians of subjects < 7 years old , and by both guardians and subjects 7 years old .
the work in this paper has been carried out in accordance with the code of ethics of the world medical association ( declaration of helsinki ) .
fasting peripheral blood samples ( 5 ml ) were collected in edta tubes , and genomic dna was extracted using the centrifugation column method with the whole blood genomic dna extraction kit ( promega ; madison , wi , usa ) according to the manufacturer 's instructions .
based on chinese han population genetics data from the database of snps ( http://www.ncbi.nlm.nih.gov/snp/ ; the us national center for biotechnology information ) , the international hapmap project ( http://www.hapmap.org/ ) , and previous studies , the drd3 snp rs6280 locus was selected for gene polymorphism analysis .
dopamine d3 receptor snp rs6280 genotyping was carried out using a taqman snp genotyping probe .
polymerase chain reaction ( pcr ) reactions ( 5 l ) contained 10 ng dna template , 2.5 l 2 taqman universal pcr master mix ( abi ) , and 0.25 l 20 snp assay ( applied biosystems , bedford , ma , usa ; including primers and fam / vic probe ) .
the reaction conditions were 95c for 10 min followed by 40 cycles of 92c for 15 s and 60c for 1 min .
pcr signals and fluorescence signals were read on an abi7900ht sequence detector ( 384-well plates ) , and then analyzed using the sds2.4 image analysis software ( abi prism , abi corp ) allelic discrimination program .
fam and vic fluorescence intensities of different alleles were analyzed to determine whether samples were homozygous wild - type , heterozygous or homozygous mutated .
spss 17.0 software ( ibm spss statistics for windows , ibm corp . , armonk , ny , usa ) was used for data analysis .
the fitness of the genotype and allele was tested using the hardy weinberg equilibrium ( hwe ) test , and polymorphism differences between the ts patient and control groups were analyzed using the chi - squared ( ) test .
the transmission disequilibrium test ( tdt ) of nuclear pedigrees was performed using plink1.07 software ( shaun purcell , usa ) , = 0.05 .
comparison of general information a total of 160 sporadic ts patients ( including 101 ts patients ) involved in pedigree studies and 59 unrelated cases underwent drd3 snp rs6280 analysis .
of the 90 normal controls , 73 were male and 17 were female , at a ratio of 4.3:1 .
there was no significant difference in the gender ratio ( p = 0.164 ) or mean age ( p = 0.927 ) between the two groups .
fitness of hardy weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in the control group ( p = 0.074 ) and ts patient group ( p = 0.582 ) , as shown by the -test .
this indicated that the data are reliable and representative for genetic association analysis [ table 1 ] .
drd3 rs6280 hardy - weinberg equilibrium test in the control and ts patient groups drd3 : dopamine d3 receptor ; ts : tourette syndrome .
comparison of dopamine d3 receptor 3 gene rs6280 genotype and allele frequencies the frequencies of drd3 rs6280 genotypes c / c , c / t , and t / t were 10.6 , 46.9 , and 42.5% , respectively , in the ts patient group , and 13.3% , 34.4% , and 52.2% , respectively , in the control group .
the frequencies of c and t alleles were 34.1% and 65.9% , respectively , in the ts patient group , and 30.6% and 69.4% , respectively , in the control group .
there were no significant differences in genotype or allele frequencies between the ts patient and control groups ( p = 0.161 and 0.423 , respectively ; table 2 ) .
comparison of genotype and allele frequencies between the ts patient and control groups ts : tourette syndrome .
comparison of general information the nuclear pedigree study included ts patients ( n = 101 ) and their biological parents ( n = 202 ) .
of the ts patients , 80 were male , and 21 were female , at a ratio of 3.8:1 .
fitness of hardy weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in both the ts patient group ( p = 0.805 ) and parents group ( p = 0.727 ) , as shown by the -test [ table 3 ] .
drd3 rs6280 hardy - weinberg equilibrium test in the ts patient and parents groups ts : tourette syndrome .
comparison of genotype and allele frequencies in the tourette syndrome patient and parents groups the frequencies of genotypes c / c , c / t , and t / t were 10.9% , 42.6% , and 46.5% , respectively , in the ts patient group , and 23.8% , 43.6% , and 32.7% , respectively , in the parents group .
the c allele frequency was 32.2% and the t allele frequency 67.8% in both groups [ table 4 ] .
comparison of genotype and allele frequencies in the ts patient and parents groups the number in brackets refers to the frequency ( % ) .
transmission disequilibrium test analysis revealed no transmission disequilibrium in the drd3 rs6280 snp among ts nuclear pedigrees , indicating that this locus is not associated with ts ( p = 1 ; table 5 ) .
tdt analysis in ts pedigree or : odds ratio ; ci : confidence interval ; ts : tourette syndrome ; tdt : transmission disequilibrium test .
comparison of general information a total of 160 sporadic ts patients ( including 101 ts patients ) involved in pedigree studies and 59 unrelated cases underwent drd3 snp rs6280 analysis .
of the 90 normal controls , 73 were male and 17 were female , at a ratio of 4.3:1 .
there was no significant difference in the gender ratio ( p = 0.164 ) or mean age ( p = 0.927 ) between the two groups .
fitness of hardy weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in the control group ( p = 0.074 ) and ts patient group ( p = 0.582 ) , as shown by the -test .
this indicated that the data are reliable and representative for genetic association analysis [ table 1 ] .
drd3 rs6280 hardy - weinberg equilibrium test in the control and ts patient groups drd3 : dopamine d3 receptor ; ts : tourette syndrome .
comparison of dopamine d3 receptor 3 gene rs6280 genotype and allele frequencies the frequencies of drd3 rs6280 genotypes c / c , c / t , and t / t were 10.6 , 46.9 , and 42.5% , respectively , in the ts patient group , and 13.3% , 34.4% , and 52.2% , respectively , in the control group .
the frequencies of c and t alleles were 34.1% and 65.9% , respectively , in the ts patient group , and 30.6% and 69.4% , respectively , in the control group .
there were no significant differences in genotype or allele frequencies between the ts patient and control groups ( p = 0.161 and 0.423 , respectively ; table 2 ) .
comparison of genotype and allele frequencies between the ts patient and control groups ts : tourette syndrome .
comparison of general information the nuclear pedigree study included ts patients ( n = 101 ) and their biological parents ( n = 202 ) . of the ts patients ,
fitness of hardy weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in both the ts patient group ( p = 0.805 ) and parents group ( p = 0.727 ) , as shown by the -test [ table 3 ] .
drd3 rs6280 hardy - weinberg equilibrium test in the ts patient and parents groups ts : tourette syndrome .
comparison of genotype and allele frequencies in the tourette syndrome patient and parents groups the frequencies of genotypes c / c , c / t , and t / t were 10.9% , 42.6% , and 46.5% , respectively , in the ts patient group , and 23.8% , 43.6% , and 32.7% , respectively , in the parents group .
the c allele frequency was 32.2% and the t allele frequency 67.8% in both groups [ table 4 ] .
comparison of genotype and allele frequencies in the ts patient and parents groups the number in brackets refers to the frequency ( % ) .
transmission disequilibrium test analysis revealed no transmission disequilibrium in the drd3 rs6280 snp among ts nuclear pedigrees , indicating that this locus is not associated with ts ( p = 1 ; table 5 ) .
tdt analysis in ts pedigree or : odds ratio ; ci : confidence interval ; ts : tourette syndrome ; tdt : transmission disequilibrium test .
current molecular genetics methods for the detection of susceptible genes in complex diseases include linkage analyses and association studies , the latter comprising either case control or pedigree studies . case
control studies compare differences in genotype and allele frequencies at candidate loci between cases and controls , so analyze the data of all subjects .
however , they are limited in that the association analysis is affected by population genetic stratification and other factors .
therefore , many association studies are difficult to repeat in different population . for this reason , pedigree studies are often preferred .
pedigree studies effectively avoid the interference of population stratification ; however , the efficiency of tdt studies has been shown to be lower than the case control studies .
the two types of analyses can complement each other , enabling the same genetic marker to be detected by both methods with more reliable results .
dopamine d3 receptor is mainly located in the region of the brain associated with cognition and affection , and the drd3 receptor is highly expressed in the mesolimbic dopamine system including the extended amygdala .
the drd3 ser9gly polymorphism ( rs6280 ) involves a serine to glycine substitution that may affect the d3 receptor affinity .
it has been widely used in studies addressing the genetic causes of neuropsychiatric disorders , including an investigation of susceptible loci correlations with psychiatric disease .
comings et al . previously identified an association between ts and homozygosity at the drd3 gene , but this was not replicated in two studies published in the same year .
demonstrated linkage disequilibrium in drd3 polymorphism loci bali and mspi , which was not associated with ts in 16 pedigrees .
found no association between ts and drd3 in 110 nuclear pedigrees by tdt analysis , while kowalska et al .
there has not been any report about the association study of ts and drd3 gene polymorphisms in han chinese population .
this study aimed to explore the association between the drd3 gene rs6280 and ts using both case controlled study and nuclear pedigrees tdt analysis .
the normal controls were well - matched with ts patients with respect to race , region , gender , and age , thus minimizing the influence of race , geography , and population stratification on experimental results , and increasing the reliability of our findings .
we found no significant differences in genotype or allele frequencies between healthy controls and ts patients , while tdt analysis also revealed no transmission disequilibrium in nuclear pedigrees .
few efforts have directly focused on the correlation between ts and drd3 , and this study was the first to do so in a chinese han population .
our combination of a case control study and nuclear pedigrees tdt analysis produced a more reliable result , which suggests that the drd3 snp rs6280 may be not associated with ts in a han chinese population .
the main is inconsistent with research method and different study populations . at the same time
, ts is a heterogeneous disorder , often comorbidity with attention deficit hyperactivity disorder and obsessive - compulsive disorder .
our findings provide no evidence for an association between drd3 gene rs6280 and ts in the han chinese population .
it should be verified by making larger samples and making further efforts to subtype ts in future study . | background : tourette syndrome ( ts ) is a complex , heterozygous genetic disorder .
the number of molecular genetic studies have investigated several candidate genes , particularly those implicated in the dopamine system .
the dopamine d3 receptor ( drd3 ) gene has been considered as a candidate gene in ts .
there was not any report about the association study of ts and drd3 gene in han chinese population .
we combined a case control genetic association analysis and nuclear pedigrees transmission disequilibrium test ( tdt ) analysis to investigate the association between drd3 gene rs6280 single nucleotide polymorphisms ( snps ) and ts in a han chinese population.methods:a total of 160 ts patients was diagnosed by the diagnostic criteria of the diagnostic and statistical manual of mental disorders , fourth edition .
the drd3 gene rs6280 snps were genotyped by taqman snp genotyping assay technique in all subjects .
we used a case control genetic association analysis to compare the difference in genotype and allele frequencies between 160 ts patients and 90 healthy controls . at the same time
, we used tdt analysis to identify the drd3 gene rs6280 transmission disequilibrium among 101 nuclear pedigrees.results:the genotype and allele frequency of drd3 gene rs6280 snps had no statistical difference between control group ( 90 ) and ts group ( 160 ) ( 2 = 3.647 , p = 0.161 ; 2 = 0.643 , p = 0.423 ) using chi - squared test .
at the basis of the 101 nuclear pedigrees , tdt analysis showed no transmission disequilibrium of drd3 gene rs6280 snps ( 2 = 0 ; p = 1).conclusions : our findings provide no evidence for an association between drd3 gene rs6280 and ts in the han chinese population . |
perforation of acalculous gallbladder in association with enteric fever is extremely rare , especially in children .
we report a rare case of enteric perforation of acalculous gallbladder in a 14-year - old boy , who presented with acute abdomen and responded very well after immediate surgical intervention .
a 14-year - old boy presented with history of diffuse abdominal pain since three days and not passing flatus and motion since 2 days .
abdominal examination revealed abdominal distension , muscle guarding , generalized abdominal tenderness and sluggish bowel sounds .
haemoglobin was 8 g / dl , platelets count was 96,000/l and wbc count was 1600/l ( neutrophil 64% , lymphocyte 28% ) .
peripheral smear showed normocytic , normochromic red blood cells with mild anisocytosis , severe leukopenia and mild thrombocytopenia .
an erect abdominal radiograph revealed multiple air - fluid levels and there was no free gas under the diaphragm .
abdominal ultrasonography demonstrated hepatosplenomegaly and gross ascites with fine internal echoes , suggestive of perforation .
peritoneal cavity was containing 2 l of bilious fluid and bowel loops were appeared to be normal . on further exploration
, gallbladder appeared to be grossly inflamed and there was a single perforation of around 1 cm in size at the fundus ( figs . 1 and 2 ) .
we looked carefully for gallstones in gallbladder , cystic duct and peritoneal cavity but not found .
post - operatively , intra - venous ceftriaxone and gentamicin were administered for 7 days .
there was an uneventful recovery and patient was discharged on 9th post - operative day .
culture of bile and peritoneal fluid were positive for salmonella typhi and escherichia coli respectively .
enteric fever is caused by salmonella typhi , s. paratyphi a , and rarely by s. paratyphi b and c. it is common in developing countries due to poor water quality and sanitation .
it is an acute systemic disease consisting of fever , malaise , transient rash , hepatosplenomegaly , abdominal discomfort and pancytopenia .
it is an intracellular infection involving the reticuloendothelial system , particularly bone marrow , liver and spleen .
bone marrow examination may show marked phagocytosis of platelets , leukocytes , and red blood cells
reported 2 cases of gallbladder perforation due to enteric fever in two boys aged 13 years and 16 years .
our patient was from tropic and was having fever , abdominal discomfort , anaemia , leukopenia , hepatosplenomegaly and widal positivity , all suggestive of enteric infection .
roslyn et al . observed that gallbladder perforation is more common in older age group . in elderly , perforation usually occurs in setting of cholelithiasis associated with obstruction leading to raised intra luminal pressure .
perforation appeared to be result of compromised blood supply and least vascularised fundus is the most common site .
in contrast , acalculous cholecystitis without obstruction is unlikely to result in perforation . in young patients ,
perforation is often associated with acute acalculous cholecystitis due to acute infections like pneumonia , viral influenza and enteric fever .
perforation in such cases is likely to be result of intense inflammation coupled with acute infection and existence of an immuno - compromised state leading to uncontrolled infection and thrombosis of the blood vessels .
our patient probably developed gallbladder perforation due to ischaemia of gallbladder wall due to acute intense inflammation .
it thought to occur more frequently in the elderly due to the loss of visceral fat and elasticity with older age , thus permitting the gallbladder to hang freely .
cholecystectomy is treatment of choice in such cases and provides good result . here , patient showed rapid recovery following cholecystectomy . in our case , laparoscopy was not performed due to unavailability of it in our emergency set - up .
fluoroquinolones or ceftriaxone ( 12 g / day ) would be optimal choices in the treatment of such cases . in uncomplicated enteric fever ,
in complicated cases , antibiotics are given for 714 days depending on condition of patient . in our case , antibiotics were given for 7 days .
our case is unusual because patient was a child , with no prior history suggestive of gallbladder disease , had no known medical co - morbidity and showed absence of gall stones on ultrasound and surgery .
gallbladder perforation secondary to enteric fever requires a high degree of clinical suspicion , especially in this age group . in typhoid endemic region , it should be considered as a differential diagnosis in patient presenting with a history of prolonged fever and signs of peritonitis .
early diagnosis and immediate surgical intervention are very important in reducing the morbidity and mortality associated with this condition .
written informed consent was obtained from the patient 's guardian for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
written informed consent was obtained from the patient 's guardian for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
| introductiongallbladder perforation is a rare complication of acute calculous cholecystitis in adults .
perforation of gallbladder due to enteric fever is extremely rare condition .
pre - operative diagnosis is rarely made and mortality is high.presentation of casewe report a case of acalculous gallbladder perforation following enteric fever in a 14-year - old boy , who presented as acute abdomen and responded very well after emergency laparotomy and cholecystectomy.discussionenteric fever is common in tropics and a common cause of bowel perforation .
acute cholecystitis is a rare complication of typhoid and gallbladder perforation is extremely rare complication . ultrasound and ct lack specificity to detect gallbladder perforation .
diagnosis is usually made intra - operatively .
cholecystectomy is treatment of choice in such cases and provides good result.conclusiongallbladder perforation secondary to enteric fever requires a high degree of clinical suspicion . in typhoid endemic region , it should be considered as a differential diagnosis in patient presenting with a history of prolonged fever and signs of peritonitis .
early diagnosis and immediate surgical intervention are very important in reducing the morbidity and mortality .
cholecystectomy is the choice with a good outcome . |
co - incidental aneurysms are detected almost seven times more frequently in patients with pituitary adenomas ( pa ) than in patients with other brain tumors2,12,19 ) .
however , the vast majority of these aneurysms are located outside the sellar - region .
aneurysms of the cavernous or supraclinoid carotid that encroach into the pa as in the present case are very rare .
the prevalence of sellar - region 's aneurysm among others is 1 - 2%14 ) .
some possible treatment options include simultaneous microsurgical managements ( frontopterional or supraorbital approach ) , transsphenoidal microsurgery after clipping or coil embolization , and two stage procedures combined with radiotherapy or radiosurgery ( residual mass)2,13,14,19 ) .
we report a patient with an incidental superior hypophygeal artery aneurysm embedded within a non - functional pa which was treated by transsphenoidal surgery after endovascular coil embolization .
the results of the neuro - ophalmologiclal examination revealed bitemporal hemianopsia . magnetic resonance ( mr )
however , the axial or coronal mr images presented a flow void in the medial portion of the right internal carotid artery ( ica ) ( fig .
these mr images raised the suspicion of a coexisting intrasellar aneurysm before the planned transsphenoidal procedure . consequently , 3-dementional computed tomography ( ct ) angiogram demonstrated the opthalmic segment of the right distal ica ( fig .
four vessels angiogram confirmed a right superior hypophygeal artery aneurysm and the saccular aneurysmal sac that had a 4.13 mm neck , 6.27 mm width , and 3.99 mm height ( fig .
first , we treated the aneurysm by coil embolization with the goal of maintaining the patency of the ica ( fig .
two months later , she underwent surgery via the transsphenoidal microsurgical approach for the removal of the tumor .
we carefully dissected the tumoral portion surrounding the coil - treated aneurysm and a total resection of the tumor was successfully achieved .
mr at 8 month follow - up after surgery demonstrated complete obliteration of the aneurysm and no sign of regrowth of the adenoma ( fig .
the incidence of intracranial aneurysm among patients with pa is 0.5% , based on the results obtained from 5762 autopsies6 ) .
since then , many studies have reported a 0.5 - 7.4% incidence of intracranial aneurysm among patients with pa , with a general consensus that this association is higher than the prevalence of intracranial aneurysm among the general patient population7,11,17 ) .
in addition , this association is generally thought to be higher in patients with pa compared with other brain tumors , although the manner in which a pa contributes to the formation of an intracranial aneurysm remains unclear5,17 ) .
al.9 ) have reported that coexistence of intracranial aneurysm was detected in 18 of 800 patients with pa ( 2.3% ) .
and then , the intracranial aneurysm in patients with pa were located in the internal carotid artery in 9 patients ( 50% ) , the middle cerebral artery in 6 patients ( 33.3% ) , the anterior cerebral artery in 2 patients ( 11.1% ) , and the vertebrobasilar artery in 1 patient ( 5.6% ) .
hanak et al.4 ) have conducted a pubmed ( national library of medicine ) literature review to identify all studies reporting non - iatrogenic aneurysms with intrasellar extension , as confirmed by ct or mr imaging and angiography .
intrasellar aneurysms were reported in functional pa such as growth - hormone pa and prolactinoma9,12,14,18 ) .
but , the coexistence of a superior hypophygeal aneurysm and a non - functional pituitary adenoma was reported in one case so far19 ) . direct infiltration by the tumor , and increased tension or blood flow in vessels supplying the tumor have been suggested as mechanisms for this coexistence of intracranial aneurysm and pa12 ) .
approximately 50% of these patients have acromegaly , suggesting that high gh and igf-1 levels or their biological effects may be implicated in the genesis of aneurysms .
a high level of igf-1 induces artery dilation , atherosclerotic and degenerative changes of the artery walls of the circle of willis , tumor invasion and tumor - directed neovessels1,9 ) .
these intrasellar aneurysms mimicking pituitary tumors are usually asymptomatic , although they can sometimes present with hypopituitarism ( fewer than 40 cases in the literature ) .
nevertheless , different clinical presentations may occur such as pituitary apoplexy or fatal epistaxis as a result of aneurysmal bleeding into the adenoma .
teng et al.15 ) demonstrated that the finding of flow voids is 100% specific for aneurysms .
however , the sensitivity of the presence of flow voids on noncontrast t1-weighted imaging , postcontrast t1-weighted imaging , and t2-weighted imaging was 88% , 22% , and 88% , respectively . furthermore , olsen et al.10 ) observed that only 12 of 15 giant aneurysms ( 80% ) showed signs of intraluminal blood flow .
these results indicate that misdiagnosis of such coexistence can be possible . in our case ,
the axial or coronal mr images presented a flow void in the medial portion of the right ica .
thus , 3-dimentional ct angiogram and 4 vessels angiogram were performed and confirmed a right superior hypophyseal artery aneurysm .
incidental intracranial aneurysms located distant from the lesion are considered to have little relevance to surgical management of most patients .
however , aneurysms of the major arteries adjacent to pituitary and suprasellar tumors are additional hazards to surgical treatment . previous knowledge of the presence and anatomy of such aneurysms is important during separation of the tumor capsule from major vessels , or removal of an intracavernous extension of a tumor lying close to the carotid artery3,7 ) .
treatment of each lesion becomes more challenging when the aneurysm lies inside the sella turcica .
endovascular coil placement is an effective treatment option if it is performed before resection of the adenoma , particularly in locations such as the cavernous carotid artery or the paraclinoid segment .
transsphenoidal microsurgical removal of the adenoma can still be performed safely in a subsequent surgical setting , but avoidance of direct contact with the aneurysm is suggested14,19 ) . the size and shape of
if the tumor is smaller than a type a suprasellar ( hardy classification ) , endovascular coil embolization followed by transsphenoidal microsurgical removal of the pa may be feasible and beneficial18 ) .
if it is not possible to achieve a total resection of pa without manipulating the aneurysm via the transsphenoidal route , simultaneous microsurgical management may be a better treatment option2 ) .
in addition , adjuvant radiotherapy or radiosurgery will be considered if recurrent tumor is found during follow up8 ) . in our case , we treated firstly the aneurysm by coil embolization and pa by the transsphenoidal microsurgical approach two months later .
we report a patient with an incidental superior hypophygeal artery aneurysm embedded within a non - functional pa .
therefore , careful evaluation of pre - operative imaging is necessary , especially in those with atypical symptoms such as our patient .
prior treatment of the aneurysm is advisable to avoid catastrophic bleeds during transsphenoidal microsurgical removal of the adenoma . | intra - cranial aneurysm can be incidental findings in patients with pituitary adenomas , and are usually located outside the pituitary region .
however , the coexistence of intrasellar ( not intracranial ) aneurysms with pituitary adenomas is extremely rare .
we report a patient with an incidental superior hypophygeal aneurysm embedded within a non - functional pituitary adenoma which was treated by transsphenoidal surgery after endovascular coil embolization . |
an operating microscope ( om ) has been considered as the mainstay in modern neurosurgery .
it produces magnified images on the retina stereoscopically while providing detailed views of surgical anatomy . during the last two decades
, neuroendoscopes are increasingly used in neurosurgery for intraventricular lesions , spine , hematoma and predominantly for trans - sphenoidal skull base surgery . an endoscope is sometimes used as an adjunct to microsurgery with om .
. an endoscope can be useful by means of providing a better overview of the operating field and also revealing detailed structures with fine illumination .
surgeons visualize the operative field from a display connected to the camera with usage of an endoscope . on the other hand , compared with om , the target is viewed only through right and left eyepieces . in 2008 , an
exoscope system i.e. the video telescope operating monitor ( vitom ) was introduced as an alternative to both om and endoscope .
the exoscope serves for observing and illuminating an object field on a patient from a position set apart from the patient s body .
it enables surgeons to complete the operation under assistance by visualizing magnified images with high resolution on a display4,5 ) .
therefore stereopsis was required to improve hand and eye coordination for microsurgical procedures with usage of the exoscope .
subsequently novel three - dimensional and high - definition operating scopes have been developed according to recent high - tech innovations in a digital surgical technology .
the standard neuroendoscope can only provide a short focal distance of approximately 3 to 20 mm .
therefore , vitom allows abundant space to insert and manipulate the instruments according to this specification ( table 1 ) .
usually the vitom is placed at regular interval of 25 cm from the operative field at first .
an illustrative case of spinal lipoma surgery with usage of vitom was shown on fig .
the main manipulations in this surgery were as follows ; drilling the vertebral arches , cutting dura and arachnoid membrane with scissors , making suspending stitches , separating adherent arachnoid , resection of the lipoma along the white plane , forming circular arc by sawing the pia matter to make a neural cord and closure of dura at last .
these operative techniques were completed under 2d motion images with a visual perception through dynamic cue and stereoscopically viewing corresponding to the motion parallax . but lacking the stereoscopic vision made it difficult to manipulate required hand - eye coordination such as taking hold the needle and stitch up .
according to my clinical experiences , three - dimensional ( 3d ) motion images must be necessary in a case with complete resection of deep seated lesions or vascular anastomosis for exoscopic surgery .
the emphasizing advantages of exoscope were regarded as the wide field of view ( fov ) and deep focus .
vitom provides the ability to maintain 35 mm depth of field ( dof ) under 50 mm fov .
it minimized the need for repositioning and refocusing during the procedure . on the other hand ,
only 50 mm fov is possible under minimum wd , however , less than 20 mm fov is easily available under 200 mm wd with om ( table 1 ) . from this point of view
, further refinements in high resolution imaging under strong magnification , such as digital zooming would be a welcome to the vitom .
in the field of laparoscopic surgery , 3d laparoscopes with various mechanical designs are currently used successfully .
those prevent signs of fatigue while shortening the operating time and facilitate various procedures inside the body , especially in performing precise suturing .
3d endoscope provides enhancing subject depth perception and spatial orientation . according to the recent reports , 3d images improve surgical dexterity in the field of neuroendoscope3,10 ) .
for example , clinical application of 3d - neuroendoscope for trans - sphenoidal surgery is regarded as an effective assistive technique and supports the feasibility and safety9,10 ) .
surgeons sometimes observe an intracranial object field from a position set apart from the sphenoid sinus during this procedure .
vsiii system ( visionsense , philadelphia , pa , usa ) is one of the valuable one .
it is based on insect - eye technology , which involves the incorporation of a single objective lens that splits light along two paths , reconstructing a 3d image9 ) . on the other hand ,
it incorporates information from two distinctive perspectives to render a single 3d view , similar to human vision with binocular depth perception .
exoscopic microsurgery with usage of the einstein vision , 3d and high - definition ( hd ) laparoscope system which is designed based on dual optical technology .
they concluded that the image quality was found to equal to that of the om , but suitability for minimally invasive neurosurgical operations is inadequate .
yoshimoto et al.10 ) reported their clinical experience of endonasal surgery with usage of dual optical channel 3d endoscope ( developed by shinko optical corporation and commercialized by machida endoscope corporation , tokyo , japan ) and compared stereoscopic images with vsiii system . according to that ,
the stereoscopic effect of dual optical channel 3d endoscope is stronger . in other words ,
dual optical channel 3d endoscope exhibits greater magnitude of binocular disparity , which is translated into the sense of extrusion and depth of structures .
in 2014 , a novel 3d and hd flexible scope called 3d - eye - flex was developed to visualize the operative field as a 3d digital image and support more accurate microsurgery6 ) .
it was introduced with an enhanced 3d - hd laparo - thoraco videoscope system ( ltf- y009 , olympus medical systems ) .
3d - eye - flex has two miniature - sized charge coupled device ( ccd ) chips at the distal end of the scope .
consequently , regardless of rigid or flexible , placing the imaging device at the tip has made on site processing of images without interruption possible , while maintaining consistent image quality .
a 15 mm - long rigid 3d and hd scope with a 15 mm outer diameter is attached to a 615 mm - long flexible bellows .
the focus distance , which is the same as the wd , is 18100 mm .
the illumination is not as bright as that of om , however , the ccds help to enhance the image . a circular polarizing filter system is used for the 3d display to deliver life - like images ( fig .
exoscope , an alternative to om , by means of capturing the 3d magnified images using digital signals from the outside of the body .
the wide angle of view and large dof inherent in the endoscope should be emphasized as advantages just like vitom system .
the large dof of 3d - eye - flex drastically minimizes the need for repositioning and refocusing during a surgical procedure .
moreover , neurosurgeons feel more comfortable when using stereoscopic vision , especially in cases of deep seated lesions . on the other hand , placing the scope close to the operative area should be required for magnifying objects .
it is more likely to be stained by blood or bone dust , fogging the lens and narrowing operative space .
as described above , recently developed operative scopes visualize the operative field as digital images and make it possible to verify the images according to application .
for example , narrow - band imaging ( nbi ) while changing the light wavelength , i.e. autofluorescence , is easily applicable with 3d - eye - flex , and can be integrated with the information obtained from white light7 ) .
an optical color separation filter narrows the bandwidth for spectral transmittance , lets only two narrow wavelengths through .
these two specific wavelengths are strongly absorbed by hemoglobin and allow a better understanding of superficial and subependymal vasculature in the ventricle ( fig .
4 ) . in another example , a fluorescent dye injected into the body leaks out of the tumor blood vessels due to the disrupted blood - brain barrier .
it is excited with an ordinary light wavelength and the yellow color - stained tumor is visible even to the naked eye . with further refinement , an ultra - high magnified image captured by endo - cytoscopy may be possible .
currently these images of the operative field are projected onto a display adjacent to navigation images or functional images .
it may be coregistered on the identical display in the near future . in state - of - the - art neurosurgery ,
neurosurgeons could highly benefit from a new surgeon s eye that visualizes the operative field with integration of the medical information received by the senses and interpreted by surgeon s brain on demand .
the novel operative scopes including exoscope system have potential for being developed to this new application
. it will set the stage for the next generation in digital image based neurosurgery . | an exoscope , high - definition video telescope operating monitor system to perform microsurgery has recently been proposed an alternative to the operating microscope .
it enables surgeons to complete the operation assistance by visualizing magnified images on a display .
the strong points of exoscope are the wide field of view and deep focus .
it minimized the need for repositioning and refocusing during the procedure . on the other hand ,
limitation of magnifying object was an emphasizing weak point .
the procedures are performed under 2d motion images with a visual perception through dynamic cue and stereoscopically viewing corresponding to the motion parallax .
nevertheless , stereopsis is required to improve hand and eye coordination for high precision works .
consequently novel 3d high - definition operating scopes with various mechanical designs have been developed according to recent high - tech innovations in a digital surgical technology .
it will set the stage for the next generation in digital image based neurosurgery . |
evidence that continuing professional development impacts positively on patient and families outcomes is necessary to sustain an on - going investment in learning activities . in order to optimize
the opportunities afforded by emerging web - based technology rural nurses need to develop and maintain their computer competencies .
further investigation of the impact of specialist clinical placements on rural nurses palliative care capabilities is also indicated .
medication is the main component of nurses function and is a major part of patients care and treatment process .
there are annually thousands of medication errors among physicians and para medicine personnel reported in the united state .
unfortunately , there is a catastrophic statistic on these errors , which has remained not reported .
these errors may be due to function of physicians , pharmacists , medication technicians , nurses or midwives .
medication errors may occur in any step of prescription and drug distribution process as prescribing medication is a sophisticated process and needs knowledge , decision making and proper function of the staffs working in various hospital wards .
their direct outcomes threaten patients life and increase costs while their indirect outcomes include nurses occupational problems and reduction of trust to them as well as their diminished function .
based on research , the burden of medication errors in national health services in england accounts to an annual 500 million dollars in addition to prolonged hospitalization days .
nurses spend about 40% of their time on preparation of drugs and medication so , they play a highlighted role in patients medication process . based on research , medication errors originate from physicians in 39% of the cases , from nurses in 38% and from pharmacists in 23% of cases .
, in a study on medication errors resulting from drug preparation and administration in internal wards , concluded that most of errors are resulted from nurses acknowledge of medication orders and their method of application . in an analysis of 469 life threatening errors between 1993 and 1998 in usa ,
human factors were reported to have the most efficient role , mostly due to a defect in practice and knowledge ( 0.44 ) .
the their study found out that nurses face problems due to lack of attention to prescribed medications dosages , ordered by a physician , wrong dosage calculation ( exchange ) and not application of their theoretical knowledge in clinical setting .
therefore , a comprehensive action is needed to solve this problem . in studies conducted in iran , more attention of authorities and nursing managers
it seems that holding educational courses to empower nurses scientific knowledge and motivation and making it more beneficial for the patients , education process modification through encouragement of nurses to report their medication errors and preparation of processes for physicians encouragement to order medication more readably in clinical setting can have a more positive and efficient outcomes .
numerous studies , conducted on the effect of continuing education on nurses efficiency , self - confidence , knowledge and skills , all show positive results so that several international studies report the effect of continuing education on improvement of cares quality .
the results of research in usa , canada , australia and england have shown the positive effect of education on quality of nursing care as well as the importance of educational programs and their effect on nurses knowledge , attitude and experimental and scientific skills .
the researcher had already determined these needs in her study on needs assessment of nurses working in psychiatric wards of hospitals in isfahan in 2011 and her obtained results showed that the most important needs of working nurses in these wards were in association with medical and nonmedical interventions including familiarization with side - effects of drugs , management of side - effects , medication indications and necessary cares at the time of medication .
with regard to above - mentioned issues and determination of nurses educational needs in relation with medical and nonmedical interventions , there is not any medical intervention workshop in iran , so we need to assess the effect of this type of program .
this study aimed to define the efficiency of an educational workshop on medical and nonmedical interventions to promote the knowledge and attitude of nurses working in psychiatric wards of educational hospitals in isfahan until 3 months after holding the workshop .
it has been coordinated to the nursing managers in noor and farabi hospitals for workshop conduction .
study population comprised all nurse working in psychiatric wards of two educational hospitals affiliated to isfahan university of medical sciences ( nour and farabi ) in 20122013 .
a total of 64 nurses ( as employees , staffs on a contract and casual staffs ) participated in the study .
inclusion criteria were at least having a bs degree in nursing and working in a psychiatric ward as an employee , on a contract or a casual staff as well as all nurses who worked part time or did overtime in the ward .
data were collected by a researcher made questionnaire which contained three sections of employment and personal characteristics , knowledge ( including 17 multiple choice questions ) and investigation of attitude ( containing 14 likert 's scale question on about psychiatry drugs indication , their side - effects , nursing care and the management of drugs side - effects ) . employment and personal characteristics were recorded and knowledge and attitude were assessed by questionnaire . whose reliability and validity were confirmed before data collection .
validity of the questionnaire was made by consideration of scientific references , and was confirmed by some academic members .
the educational method employed in the present study was lecture , question and answer and group discussion .
it should be noted that the researchers conducted the study with regard to the results obtained from the needs assessment of nurses working in psychiatric wards of hospitals affiliated to isfahan medical sciences university in 2012 .
educational protocol and related workshops were designed through formation of a scientific committee ( including some of psychiatry professors and nursing educators of isfahan university of medical sciences as well as nursing managers and educational supervisors ) and discussion about obtained educational needs .
the outlines of the educational workshop contained six sections : the generic name of drugs , recognition of dosage and forms , indication and diagnose side effects , electroconvulsive therapy ( ect ) indication and side effects , caring for drugs prescription and the management of drugs side - effects .
nurses level of knowledge and attitude was investigated by the questionnaire before beginnings of the workshop , at its end and 3 months after holding the educational workshop . to have the highest possible number of attendants in the workshop , the workshop was held by academic members of the related university in two different times and in two various hospitals ( nour and farabi ) . at the end of workshop
, the attendants were given workshop educational materials in form of a cd and the credit of continuing education course .
repeated measure anova with control of some baseline and confounding factors as well as bonferroni test were used for data analysis .
sixty - four nurses attended the present study of whom 29.7% were male and 70.3% were female .
the highest frequency of employment condition was for casual staffs ( 87.5% ) and permanent staffs ( 12.5% ) .
the results showed a significant increase in nurses knowledge mean scores in relation with medical and nonmedical immediately after and 3 months after education ( p < 0.001 ) so that it increased from 61.5 14.8 ( before education ) to 81.7 1.8 ( immediately after ) and to 79.9 13.8 ( 3 months after ) [ table 1 ] .
mean scores of nurses knowledge and attitude before , immediately after and 3 months after interventions furthermore , there were a significant difference between mean scores of nurses attitude about medical and nonmedical interventions before , immediately after and 3 months after education ( p < 0.009 ) [ table 1 ] .
based on table 2 , comparison of nurses knowledge mean scores of three stages ( before education and immediately after ) , ( before education and 3 months after ) and ( immediately after education and 3 months after ) , there were significant association between before education and immediately after it .
also , there were significant relation between mean scores nurses knowledge before education and after 3 months .
comparison of nurses knowledge concerning medical and nonmedical interventions nurses knowledge and attitude as adjusted variables , before education and immediately after ) , ( before education and 3 months after ) and ( immediately after education and 3 months after ) was evaluated by the bonferroni correction . based on table 3 ,
the mean of attitude scores showed a significant difference between immediately after education and 3 months after it .
comparison of nurses attitude concerning medical and nonmedical interventions based on table 4 , the results showed a significant increase in nurses knowledge about dangerous side - effects of clozapine , side - effects of new antidepressant drugs and monoamine oxidase ( mao ) inhibitors , antidepressant drugs groups , extra pyramidal side effects , side - effects of selective serotonin reuptake inhibitors ( ssris ) and lithium and drug interactions with ect .
distribution of nurses correct answers in response to medical and nonmedical interventions question before , immediately after and 3 months after intervention the obtained results showed that 86.3% of nurse were either
satisfied with the educational course held for them and believed existence of such a course led to their scientific information improvement .
this study aimed to investigate the efficiency of educational intervention on the levels of nurses knowledge and attitude in psychiatric wards about medicational and nonmedicational interventions .
the obtained results showed the effect of a medicational and nonmedicational interventional educational workshop on improvement of knowledge level of nurses working in psychiatric wards immediately after and 3 months after education in 15 items .
also , only one item ( recognition hypotension orthostatic ) , the knowledge was decreased however it was not statistically significant .
these results is consistent with study of pasyar et al . on the effect of continuing education concerning medication indications on promotion of nurses knowledge and attitude .
this also confirms the high effect of education on increase of the knowledge in treatment personnel , and consequently , on promotion of patients health and relatively improvement of services quality and nurses job satisfaction .
this method seems to have a high effect on preservation of information until 3 months after intervention . in a study by abbaszadeh et al . in kerman ,
iran , suggests that continuing education program promote knowledge , attitude and performance of nurses on documentation . in a study conducted in northern italian university on effectiveness of a clinical skills workshop for drug - dosage calculation with participation of 77 nursing students
, the results showed that clinical skills workshop increased drug - dosage calculation as well as students understanding of mathematical calculation , which is consistent with the present study .
in evaluation of the effect of educational workshop on nurses knowledge concerning medicational and nonmedicational interventions , the results showed a significant increase in nurses knowledge about dangerous side effects of clozapine , side effects of new anti - depressant drugs and mao inhibitors , antidepressant drugs groups , extrapyramidal side effects , side effects of ssris and lithium and drug intractions with ect .
the results of the present study seemed to have met most of the nurses needs revealed in the nurses needs assessment , conducted several months before holding the educational workshop by the researcher , including
familiarization with drugs side effects , management of side effects , and necessary nursing cares in drugs consumption .
research showed that nurses staffs education in hospitals about paying a close attention to medication on precise time leads to improvement of medication management and quality of patients care as well as reduction of side - effects , which is consistent with the present study .
it seems that current continuing education programs held for nurses have not been adequately successful in increase of nurses knowledge and attitude due to lack of purposefulness of educations , lack of needs assessments and the type of education ( no workshop held ) .
research showed that if educational programs are purposefully held based on nurses needs and in form of workshops , they can significantly affect nurses knowledge , attitude and skills , something we observed in the present study .
another variable investigated in the present study was determination of nurses attitude for which the results showed the effect of educational intervention on the attitude of nurses working in psychiatric wards immediately after and 3 months after education .
dadgari et al . showed that educational workshop leads to improvement of knowledge , attitude and practice of nurses in critical care units .
mehdipur showed that educational classes result in an increase in nurses attitude and their more preparation to educate the patients , which is in line with the present study .
several studies showed weakness of the nursing students and personnel working in hospitals about psychiatric medications , insufficient skills of correct medication due to brand name and labels similarities as well as wrong dosage calculations . on the other hand , other studies indicated the effect of educational programs on improvement of medicational care quality . therefore , nursing personnel education is going ahead with a rapid pace toward professionalism and employees empowerment parallel to coordination with other medical professions , and curriculum education and just attaining a university certificate can not be a license for work during an employee 's working life any more .
in fact , education in medical groups does not end in university but continues during professional activities .
brady and hyde believes nurses knowledge and preparation for continuing education is essential . in this direction , detection of educational needs and provision of facilities
therefore , the role of continuing education should be considered as a basic need for nurses adaptation with rapid increasing scientific changes , and determination of educational priorities based on needs assessment as the main step in educational planning .
nurses are frequently exposed to new advancements during their professional carrier and should update their knowledge about drugs specially the generic names of medications , dosage and medication types , and drugs side effects and contraindications . according to the results of the present study , a proper background for nurses attendance in educational programs based on needs assessment should be prepared for their professional promotion .
our obtained results showed that holding an educational workshop was effective on nurses knowledge and attitude toward medical and nonmedical interventions .
results of this study confirm that purposeful continuing education is essential for nurses to promote the quality of nursing cares . with regard to the role of nurses in medical interventions and the necessity of their being up - to - date with medical information as one of their necessary skills to reduce medication errors and the most important and life - threatening treatment complications , nurses should be provided with the required conditions for attendance in educational programs based on their professional needs .
the researchers faced problems and limitations in conducting this study of which the most important ones were the limited population of participants , and short length of education period and follow - up .
the researchers faced problems and limitations in conducting this study of which the most important ones were the limited population of participants , and short length of education period and follow - up .
| background : the medical and nonmedical care of patients is the necessary skills in nursing profession . that it needs proper knowledge and attitude .
hence , it is important to promote nurses knowledge and attitude by education based on need assessment .
this study aimed to define the efficacy of a medical and nonmedical intervention educational workshop on nurses knowledge and attitude until 3 months after holding the workshop in psychiatric wards of educational hospitals in isfahan.materials and methods : this is a quasi - experimental study .
the study population comprised all nurses working in psychiatric wards of nour and farabi hospitals ( 64 ) in isfahan in 2012 .
an educational workshop was held through educational sessions in the form of lectures and group discussion in two above - mentioned hospitals .
nurses level of knowledge and attitude were investigated by a researcher made questionnaire before , immediately after and 3 months after intervention .
data were analyzed by descriptive statistical tests of repeated measure anova and bonferroni.results:a significant increase was observed in mean scores of nurses knowledge immediately after and 3 months after education compared to before education ( p < 0.001 ) .
also , the mean of attitude score in 3 phases have significant different ( p < 0.009 ) .
nurses have the high satisfaction ( 86.3% ) of need assessment based education workshop .
moreover , it is effective in the science information revival of nurses.conclusion:educational sessions notably affected the promotion of nurses knowledge and attitude . with regard to nurses satisfaction with the workshop which was held , designing and organizing educational workshops based on constant needs assessment is suggested for the promotion of nursing cares . |
creativity is a very important psychological phenomenon that has attracted increased research interest in the cognitive neurosciences . at the same time
, creativity is an extremely complex phenomenon that renders such research rather more difficult than studying a more basic cognitive process , such as attention or memory . because of these difficulties
, the empirical research does not always generate consistent results . in part , these inconsistencies can be attributed to the immense variety of creativity measures .
there are far more ways of measuring creativity than there are of measuring general intelligence , for example , and these diverse methods do not even have to agree with each other .
the goal of this brief report is to survey the alternative routes to assessing creativity and to suggest an integrative approach to such assessment .
however , before we can do so , it is first necessary to define what creativity means
. it would be most unwise to start measuring something before we know what we are trying to measure .
unfortunately , researchers have been somewhat too creative in their definitions , with over a dozen possibilities being suggested in the literature .
most investigators seem to favor a two - criterion definition : an idea or response is said to be creative if it is ( i ) novel or original ; and ( ii ) useful , adaptive , or functional .
the drawback to this definition is that it is perfectly feasible for an idea to be novel and useful without being necessarily surprising .
algorithmic solutions are of this nature . because the cognitive processes supporting algorithmic problem solving are quite unlikely to be similar to the processes supporting more heuristic problem solving , it is advisable to add a third criterion , namely , surprising or
this three - criterion definition has several repercussions , including the increased necessity of engaging in blind - variation and selective - retention ( bvsr ) processes . yet , from the standpoint of this brief note , the main implication is that creativity must be separated from both general intelligence and domain - specific expertise , neither of which can produce anything surprising because each is dedicated to converging on the single most correct response .
indeed , the convergent thinking witnessed in the application of general intelligence and domain - specific expertise is designed for different kinds of problems than for divergent thinking and other processes seen in creativity . a nice illustration is the distinction between reasonable problems that can be reasoned out step by step to home in on the solutions ( eg , anagrams and crossword puzzles ) and unreasonable problems that do not lend themselves to step - by - step thinking .
one has to sneak up on them , eg , all true insight problems ) . because solutions to unreasonable problems involve some problem restructuring ( eg , serendipitous changes in problem representation ) , such solutions tend involve a eureka or
given the foregoing definition , we then have to figure out the optimal procedures for assessing creativity it turns out that the options are , if anything , too numerous .
many researchers attempt to measure the processes presumed to be responsible for the generation of creative ideas , such as divergent thinking ( dt ) and remote associations ( rat ) .
other researchers concentrate on assessments of the creative person , most often via some personality measure , such as the creative personality scale ( cps ) of the gough adjective check list .
in addition , because individual differences in creativity strongly correlate with both the openness to experience factor in the five -factor model and the psychoticism scale of the eysenck personality inventory , these latter measures can be used as indirect predictors . taking a different tack
, other investigators will focus on the creative product , often using the consensual assessment technique ( cat ) .
for example , scores on the cps correlate positively with divergent thinking . and both openness to experience and psychoticism correlate positively with defocused attention or reduced latent inhibition , which has been identified as an important process in creative thought .
moreover , the creativity of persons can be gauged by the number of creative products or actions they have generated , identified through either self - reports or bibliographic sources .
because creative productivity is strongly associated with achieved eminence , some researchers will use expert evaluations or conspicuous awards as indicators of creativity .
such historiometric measures have been shown to have some highly desirable features , including high reliability and face validity .
implicit in the above inventory of measures is a subtle shift in the magnitude of the creativity assessed . at the lower level is everyday , psychological , or
little - c creativity , whereas at the higher level is eminent , historical , or big - c creativity . on the one hand , lower - level creativity is most often gauged using a process measure , such as the unusual uses test , or an everyday product measure , such as the cat . on the other hand
, higher - level creativity is most often measured using an eminence or productivity indicator .
another important difference is that little - c creativity is usually assessed using generic instruments that are assumed to be applicable to any domain ( eg , the rat ) , whereas big - c creativity is most often quantified via measures that are inherently domain - specific .
thus , the creative output of a scientist might be recorded by domain - specific publications and citations as well as award recognition .
the key question is whether it is possible to create a comprehensive measurement tool that does for creativity what iq tests do for intelligence .
that is , can we devise a scale that taps creativity from almost trivial problem solving to the accomplishments of creative genius and everything between , without a single hiatus ?
most desirably , this measure should be applicable to every major form of creativity rather than being tied down to a particular domain . at present , no such instrument exists , but i would like to suggest the most promising starting point for future developments : the creative achievement questionnaire or caq .
although the caq concentrates on actual achievements , these achievements are scaled from an effective zero point ( none whatsoever ; the person claims no talent or training ) through various degrees of little - c creativity ( eg , having written a poem or short story ) , and ending with domain - specific accomplishments of a very high order ( having received a national prize ) .
the caq also assesses creativity in several distinct domains , including scientific inquiry , creative writing , humor , theater and film , visual arts ( painting , sculpture ) , architectural design , music , dance , inventions , and culinary arts .
finally scores on this measure positively correlate with such person measures as openness and the cps , and with such process measures as divergent thinking ( including its components fluency , originality , and flexibility ) , and thereby taps into more than just product assessment .
the caq has already joined the inventory of creativity measures used in the cognitive neurosciences .
even so , it would appear that the next step should be an integrative battery of tests that combine the product - oriented caq with both process and person measures that would better anchor the lower end of the underlying creativity dimension .
in addition , the upper end of the scale can be further refined by introducing measures of broader impact , such as citation measures and domain - specific awards that differentiate the best from the very best . within the sciences ,
a nobel laureate dwells at a more elite level than elevation to the national academy of sciences . precisely merging these diverse assessments at opposite ends of the caq
interpolating such heterogeneous measures into a single indicator would require extremely careful calibration based on large samples of research participants who vary greatly in creativity .
complicating matters even further , the calibration of the upper end of the scale would have to be executed separately for each domain and even sub - domains .
the eminence of physicists can not be scaled in the exact same way as the eminence of psychologists .
a closely related complication concerns the transition from subjective assessments of creative achievement in the middle portion of the scale to objective assessments of creativity achievement at the upper end of the scale . on the one hand
, the caq asks respondents to self - report their products and awards , a clearly subjective judgment that might differ from one respondent to another . on the other hand , productivity , eminence , and similar historiometric measures of achievement
again largely domain - specific to learn how the former method can be made to dovetail properly with the latter method .
the difficulties aside , some kind of psychometric integration of creativity measures is required if we are ever going to be able to differentiate einstein 's brain from the brain of his less distinguished colleagues , as well as separate the brain of a competent but noneminent scientist from someone who is struggling to pass a university science course .
if we can gauge intelligence across its full population variance , we must be able to do the same for creativity . besides iq , we would possess something that might be styled cq . until we obtain a proper cq instrument , | because the cognitive neuroscientists have become increasingly interested in the phenomenon of creativity , the issue arises of how creativity is to be optimally measured . unlike intelligence , which can be assessed across the full range of intellectual ability creativity measures tend to concentrate on different sections of the overall spectrum .
after first defining creativity in terms of the three criteria of novelty , usefulness , and surprise , this article provides an overview of the available measures .
not only do these instruments vary according to whether they focus on the creative process , person , or product , but they differ regarding whether they tap into little - c versus big - c creativity ; only productivity and eminence measures reach into genius - level manifestations of the phenomenon .
the article closes by discussing whether various alternative assessment techniques can be integrated into a single measure that quantifies creativity across the full spectrum . |
diyarbakir is the largest residential area in the southeastern anatolia of turkey .
routine hbv vaccination has begun to be implemented by the ministry of health in turkey in 1998 .
the purposes of this study were to detect the levels of hbv dna in patients with hbv in 2012 , and to compare the results of the year 2002 according to age groups .
hbv dna results of patients were divided in to seven age groups ( 0 - 14 , 15 - 20 , 21 - 30 , 31 - 40 , 41 - 50 , 51 - 60 , and > 61 years ) and for comparison of hbv dna levels of 2002 and 2012 , hbv dna values in pg / ml of year 2002 were translated into iu / ml and hbv dna levels were grouped as < 5 pg / ml < 2.43 105 iu / ml , 5 - 100 pg / ml 2.43 105 - 4.86 106 iu/ ml , 101 - 2000 pg / ml 4.87 106 - 9.72 107 iu / ml , > 2000 pg / ml > 9.72 107 iu / ml 2 - 3 .
a statistically significant decrease was seen in the number of individuals in 0 - 14 age group in 2012 compared with 2002 . in 2002 the rate of individuals in 0 - 14
age group was 18.8% whereas 4.8% in 2012 .
our study was suggested that that routine hbv vaccination program , contributed to the reduced risk of hbv infection in our region . | |
the patient was male , aged 50 , 169 cm and 72 kg , and visited the hospital to undergo shoulder joint surgery with a damaged in the right rotator cuff .
although he had diabetes that was diagnosed ten years before , he did not undergo treatment for it ; however , blood sugar level had been controlled .
the patient sometimes complained of palpitations and underwent tests for that in another hospital , but no anomaly was found and he was being followed - up .
no specific findings were found in the chest radiograph or echocardiogram , and laboratory findings were normal .
vital signs just before induction and after arriving in the operation room were as follows : blood pressure of 160/90 mmhg , heart rate of 70/min , and oxygen saturation of 97% , and electrocardiography showed normal sinus rhythm .
anesthesia was induced with pentothal sodium 300 mg , fentanyl 100 g , and rocuronium bromide 50 mg , and the endotracheal tube ( inner diameter 8.0 mm ) was inserted orally . for maintenance of anesthesia , 50% n2o - o2 , sevoflurane 3 vol% , and remifentanil 0.05 g / kg / min were used . after vital signs
were stabilized , concentration of sevoflurane was adjusted to 1.5 vol% and atropine sulfate 0.5 mg was intravenously injected just before changing the patient 's position from supine to a sitting position ( beach chair position 70 - 80 ) .
one or two minutes after sitting position , heart rate was suddenly reduced from 80/min to 30/min or less , and blood pressure was 70/40 mmhg . but no electrocardiograhic changes were noted . intravenous injection of atropine sulfate 0.5 mg was immediately given , but heart rate and blood pressure did not change . after stopping the administration of remifentanil and sevoflurane , 1 : 10,000 epinephrine 1 ml ( 10 g ) was intravenously injected , and the patient was moved to the supine position .
the results of the arterial blood gas analysis at that time were within normal range . through the discussion with surgeons
detailed tests were performed a 24-hour electrocardiography and echocardiography , and the patient was suspected of having vasovagal syncope suspect .
a sufficient amount of fluid was supplied and a -sympatholytic was orally administered as the preventive treatment . following preoperative administration of anticholinergic and sedative , anesthesia was induced well with desflurane and remifentanil .
the hypotension accompanied by bradycardia in this case may appear similar to syncope ( i.e. , bjr activation ) .
bjr is overlapped by vasovagal syncope , one type of neurocardiogenic syncope , which is caused by reduction of venous return to the heart due to the decrease of peripheral arterial pressure by the neurogenic mechanism .
bjr is a reflex action that causes three signs : vasorelaxation , bradycardia , and hypotension .
lower extremity muscle contraction is necessary in maintaining venous return to the heart and blood pressure .
general anesthesia as well as epidural anesthesia inhibits such a " muscle pump " and the compensatory arterial and venous constriction .
syncope due to the phobia and pain during general induction has been reported sometimes ; however , it is relatively rare in general anesthesia .
venous congestion caused by the sitting position during shoulder joint surgery under general anesthesia brings about the decrease of intraventricular volume , and rapid preload decrease causes abnormal myocardial contraction by the hypersecretion of catecholamine .
enhanced myocardial contraction under reduced preload activates mechanical receptors of the heart , abnormally activates parasympathetic system , and brings about repression of sympathetic system . as a result
paradoxical hypotension and bradycardia can be caused by intracardiac bjr activation because of blood flow dysfunction and vasodilator due to venous blood stasis and the insufficiency of intracardiac blood that leads to vigorous cardiac contraction .
when a headup tilt test is performed , it takes approximately 12 - 24 minutes until hemodynamic changes , including the decrease of mean arterial blood pressure and heart rate begin , but it may take about one hour if the position is tilted by approximately 60 . in this case ,
severe bradycardia started immediately when the sitting position was taken about 20 minutes after induction .
there are two types of vasovagal inhibition : vasodilatory ( vd ) and cardioinhibitory ( ci ) type .
the vd type consists of a drastic reduction of arterial blood pressure without bradycardia , whereas the ci type is characterized by bradycardia associated with hypotension .
the reported frequency of bradycardia and hypotension in the sitting position during interscalene block has been 13 - 29% [ 4,9 - 11 ] .
the severity of bradycardia and hypotension varies ranging from a mild state to cardiac arrest and severe complications such as temporary loss of vision , cerebral and spinal ischemia , and ophthalmoplegia .
appropriate fluid and -sympatholytic should be intravenously injected to maintain sufficient volume of blood before bjr takes place , and preventive anticholinergic administration should be performed to repress the vasovagal reflex . moreover ,
the venous return volume should be immediately supplemented by altering position from the sitting to the supine position .
the cephalic region should be lowered and the lower extremity should be lifted up . the reduced ventricular volume caused by the orthostatic position and excessive myocardial contraction induce bjr by stimulating the intracardiac mechanical receptors . even though atropine sulfate has been used as the only drug to treat the asystole that takes place under general anesthesia , it was not effective in our case .
sympathomimetic drugs , such as ephedrine and various selective -agonists , repress venous and arterial vasorelaxation , and the rapid injection of epinephrine has been emphasized as an important factor of successive treatment . in various studies on the treatment of vasovagal syncope , it was reported that atropine sulfate had efficacy in the ci type but not the vd type , and that studies had not been conducted yet with a sufficient number of patient to make a conclusion with respect to the effectiveness of vasovagal syncope treatment .
currently , treatment methods for relapsing vasovagal syncope are divided into long - term , oral drug and pacemaking .
these treatment methods serve different purposes : drug treatment is effective in inhibiting initiation of the vasovagal reflex , whereas pacemaking can eliminate the reflex action immediately after symptom appears .
based on this case , syncope should be examined in a patient who is to undergo the shoulder joint surgery in the sitting position under general anesthesia .
if suspected , specific tests such as head - up tilt testing may be necessary .
additionally , in the case of suspected syncope by intraoperative positional change or other causes , the patient should be quickly moved to the supine position , and the selection of appropriate drugs should be taken into consideration . | the occurrence of severe hypotension and bradycardia , following placing to the beach chair position from supine during general anesthesia for repair of tendon injury of the rotator cuff of shoulder in a healthy 50 year - old man was described .
the bezold - jarisch reflex , which is known to inhibit cardiovascular reflex and composed of three kinds of symptoms such as vasodilation , bradycardia and hypotension , has been reported mainly in peripheral nerve block , and may occur during orthostasis , hypovolemia , hemorrhage , supine inferior vena cava compression in pregnancy , interscalene block for shoulder surgery in the sitting position and so on .
the bradycardia and hypotension can be more aggravated when causative elements overlaps each other .
anticholinergics and vasopressor were injected intravenously , and position of the patient was changed to the supine position immediately resulting in a normal vital signs dramatically . |
adenosine triphosphate - binding cassette transporters , such as p - glycoprotein ( pgp ) , breast cancer resistance protein and multidrug resistance protein 4 have a pivotal role in controlling brain distribution of drugs .
these transporters are expressed in the luminal membrane of brain capillary endothelial cells that form the blood brain barrier ( bbb ) and are capable of effluxing a multitude of chemically diverse compounds , often in cooperation with transporters expressed at the abluminal membrane , from brain into blood .
transporter - mediated drug drug interactions at the bbb are of great concern as they may lead to altered brain distribution of drugs and hence to severe central nervous system side effects .
studies in mice have shown that genetic knockout of pgp can result in up to 2,600% increases in brain - to - plasma ratios of drugs , which are selective pgp substrates ( e.g. , ivermectin ) . however , for drugs that are in addition to pgp recognized by other efflux transporters at the bbb much smaller increases in brain distribution will be achieved when only pgp is inhibited , as the other non - inhibited transporter(s ) may compensate the function of pgp . to better understand whether results from rodent studies translate to humans it is important to assess the effect of transporter inhibition on drug brain distribution in humans .
positron emission tomography ( pet ) imaging with radiolabelled pgp substrates , such as [ c]verapamil or [ c]n - desmethyl - loperamide , in combination with administration of pgp inhibitors has now made it possible to directly study pgp - mediated drug drug interactions at the human bbb .
two pgp inhibitors have so far been used in clinical pet studies , the immunosuppressant cyclosporine a and the non - marketed third - generation pgp inhibitor tariquidar . whereas cyclosporine a caused only small increases in brain uptake of [ c]verapamil and the use of higher doses is limited by safety concerns , tariquidar is relatively well tolerated at doses required to inhibit pgp at the bbb .
administration of tariquidar at a dose of 6 mg / kg body weight was shown to result in 129% and 223% increases in brain distribution of ( r)-[c]verapamil and [ c]n - desmethyl - loperamide , respectively , in humans . however , as these effects were smaller than those observed in rodent studies it was not clear whether complete inhibition of pgp at the human bbb had been achieved . knowing the maximum possible effect of pgp inhibition on brain distribution of a pgp substrate in humans is important to better understand the safety risks associated with pgp - mediated drug drug interactions at the human bbb and to obtain much needed scaling factors for extrapolation of data from rodents to humans . in previous studies ,
pet scans with ( r)-[c]verapamil or [ c]n - desmethyl - loperamide were performed at approximately 1 hour after completion of an intravenous tariquidar infusion .
as tariquidar plasma concentrations rapidly decline after end of infusion , we investigated in the present study the effect of continuous infusion of tariquidar for the duration of the pet scan on ( r)-[c]verapamil brain distribution .
the study was registered with eudract ( number 2012 - 005796 - 14 ) , approved by the ethics committee of the medical university of vienna , and conducted in accordance with the declaration of helsinki and its amendments . from all subjects written informed consent was obtained before enrollment into the study .
five healthy men ( mean age : 273 years , mean weight : 7914 kg ) were included into the study .
subjects were required to be free of any medication for at least 14 days before start of the study .
subjects underwent two consecutive 60-minute dynamic ( r)-[c]verapamil pet scans ( mean injected activity : 40016 mbq ) on a ge advance scanner ( general electric medical systems , milwaukee , wi , usa ) and serial arterial blood sampling via the radial artery as described previously .
one hour before start of the second pet scan an intravenous infusion of tariquidar was started at an infusion rate of 375 ml / h and maintained until the end of the scan ( mean infusion time 1233 minutes ) .
for formulation of the infusion solution , a stock solution ( 70 ml ) of 7.5 mg / ml of tariquidar free base in 20% ethanol/80% propylene glycol ( azatrius pharmaceuticals pvt ltd , london , uk ) was diluted with aqueous dextrose solution ( 5% , w / v , 805 ml ) to a final concentration of 0.6 mg tariquidar free base per ml .
three venous blood samples ( 5 ml ) were collected at the beginning , in the middle and at the end of the second pet scan to measure plasma concentrations of tariquidar by a previously described liquid chromatography tandem mass spectrometry assay .
an arterial input function was constructed by correcting total activity counts in arterial plasma for polar [ c]metabolites of ( r)-[c]verapamil as described previously .
plasma protein binding of ( r)-[c]verapamil in blood samples obtained immediately before each pet scan was determined as described before .
all subjects underwent t1-weighted 3 tesla magnetic resonance imaging on an achieva 3.0 t scanner ( philips medical systems , best , the netherlands ) .
the pet and magnetic resonance data were processed with analyze 8.0 ( biomedical imaging resource , mayo foundation ) and spm5 ( wellcome department of imaging neuroscience , ucl ) software and whole - brain gray matter time - activity curves were extracted using the hammersmith n30r83 three - dimensional maximum probability atlas of the human brain as described previously .
in addition , the pituitary gland was manually delineated on axial slices of coregistered pet and magnetic resonance data sets with pmod 3.6 software ( pmod technologies ltd , zrich , switzerland ) .
a standard 2-tissue 4-rate - constant ( 2t4k ) compartment model was fitted to the time - activity curves from 0 to 60 minutes after radiotracer injection as described previously .
differences in outcome parameters of scan 1 and 2 were tested using the wilcoxon matched pairs test ( statistica 6.1 , statsoft inc . ,
concentration - effect relationship was analyzed by nonlinear regression based on a sigmoidal model with variable slope using the prism 5.0 software ( graphpad software , la jolla , ca , usa ) .
in a previous study in which we administered tariquidar as a short infusion over 30 minutes during an ( r)-[c]verapamil pet scan we observed that ( r)-[c]verapamil brain concentrations were rising in immediate response to tariquidar infusion and were rapidly falling after the end of infusion in parallel with declining tariquidar plasma concentrations ( supplementary figure i ) .
this suggested that the pgp - inhibitory effect of tariquidar at the bbb is transient .
this is in good agreement with another study , which demonstrated that higher increases in brain uptake of [ c]n - desmethyl - loperamide can be obtained in humans when the pet scan is performed during rather than after tariquidar administration .
the transient inhibition of pgp at the bbb by tariquidar prompted us to perform in this study two consecutive ( r)-[c]verapamil pet scans , a first baseline scan and a second scan during continuous infusion of tariquidar , in analogy to a previously described pet protocol using cyclosporine a as pgp inhibitor .
we started the tariquidar infusion at 60 minutes before start of the second pet scan and maintained it throughout the pet scan , which resulted in the administration of a mean tariquidar dose of 5.91.0 mg / kg body weight . in four out of five subjects
previously known , mild adverse reactions that were most likely related to tariquidar occurred ( metallic taste in the mouth : n=3 and dizziness : n=1 ) .
mean tariquidar plasma concentration during the pet scan was 2,873517 nmol / l ( supplementary table i ) , which was 23 times higher than in our previous study .
tariquidar administration had no significant effect on the fraction of non - protein bound ( r)-[c]verapamil in plasma ( scan 1 : 0.0840.016 , scan 2 : 0.1030.016 ; p=0.11 ) , which was in good agreement with the previous results .
brain time - activity curves were approximately four times higher in scan 2 as compared with scan 1 ( figure 1a ) .
the pet data were analyzed with kinetic modelling using a 2t4k model , which was shown in previous studies to be the model of choice for modelling of [ c]verapamil pet data after pgp inhibition , to obtain vt and the transfer rate constant from plasma into brain k1 as outcome parameters of interest ( table 1 ) .
vt and k1 values were significantly increased by 27378% and 25974% , respectively , relative to baseline scans ( p=0.043 ) .
mean vt and k1 values in scan 2 were 1.7 and 1.6 times higher than mean vt and k1 values in the highest tariquidar dose group ( 8 mg / kg ) of our previous study .
interestingly , the increases in brain uptake of ( r)-[c]verapamil observed in the present study were of similar magnitude as the increases observed for [ c]verapamil in another study in non - human primates during administration of a high dose of cyclosporine a , which presumably led to complete pgp inhibition at the bbb ( mean 372% increase in [ c]verapamil k1 ) . it has been hypothesized that uptake of [ c]verapamil in the posterior pituitary gland , which has fenestrated capillaries and is therefore not protected by the bbb , may provide an estimate of brain uptake of [ c]verapamil in the absence of pgp function .
we therefore analyzed distribution of ( r)-[c]verapamil to the pituitary gland ( figures 1a and 1b ) . while vt in the pituitary gland was 5.31.7 times higher than in whole - brain gray matter in scan 1 ( p=0.043 ) , vts in whole - brain gray matter and pituitary gland were comparable and not significantly different in scan 2 , which supported the assumption that we were approaching complete inhibition of pgp at the bbb ( table 1 ) .
moreover , vt in the pituitary gland was similar in scans 1 and 2 , which suggested that distribution of ( r)-[c]verapamil to this region is not restricted by pgp ( table 1 ) .
it should be noted , however , that the pituitary gland lies in proximity of two arteries and is smaller ( mean volume of interest : 0.110.02 cm ) than the spatial resolution of our pet scanner ( 6 mm ) so that quantification of radioactivity in the pituitary gland may be affected by partial volume and/or spill - over effects .
we used the data obtained in this study and data from a previous study to extend our previously published concentration - effect curve of tariquidar for enhancement of brain uptake of ( r)-[c]verapamil .
a sigmoidal curve provided good fits of the combined data ( figure 1c ) giving an estimated half - maximum effect concentration ( ec50 ) of 2,248
nmol / l , which was higher than the previously estimated ec50 of tariquidar in humans ( 868 nmol / l ) .
the estimated maximum vt in brain ( 3.5 ) was comparable to vt in the pituitary gland in scan 2 ( 3.060.72 ) .
the ratio of nonprotein bound plasma concentration of a transporter inhibitor to its in vitro inhibitory constant ( ki ) has been proposed to predict the propensity of an inhibitor to achieve significant transporter inhibition in vivo . in our study ,
the fraction of unbound tariquidar in human plasma has been determined before as 0.00630.0021 , corresponding to unbound tariquidar plasma concentrations in our study ranging from 11 to 26
the ki of tariquidar for inhibition of transport of [ h]verapamil by human pgp is 8.5 nmol / l ( unpublished data ) giving a ratio of the unbound tariquidar plasma concentration to ki ranging from 1.3 to 3.0 .
since brain distribution of lipophilic pgp substrates , such as ( r)-[c]verapamil or [ c]n - desmethyl - loperamide , may be dependent on cerebral blood flow ( cbf ) , it has been suggested that extraction fraction ( e ) given as k1 divided by cbf may be a more suitable parameter of pgp function than k1 or vt .
however , since we did not measure cbf in the present study , we were only able to estimate e based on cbf values available from the literature ( 0.5 ml / g per minute for whole - brain gray matter ) .
normalization of k1 values in scan 2 to cbf provided e values of 0.300.04 and 0.440.12 in whole - brain gray matter and pituitary gland , respectively , which indicated that for ( r)-[c]verapamil extraction may not reach 100% even in the absence of pgp activity .
it should be noted , however , that cbf in the pituitary gland may differ from cbf in whole - brain gray matter so that our e estimate for the pituitary gland may not be accurate . in a previous study in rats a 932% increase in ( r)-[c]verapamil brain vt over baseline scans
was observed after complete inhibition of pgp at the bbb by tariquidar . according to our concentration - effect curve ( figure 1c ) ,
the estimated maximum vt in human brain is 3.5 , which allows predicting a maximum vt increase in humans of 367% .
this is in good agreement with the prediction made in another study for [ c]verapamil ( + 400% ) .
this indicates that for ( r)-[c]verapamil the scaling factor between rats and humans for vt increase after complete pgp inhibition is 2.5 .
it is , however , not known whether this scaling factor will also apply to other substrate - inhibitor combinations than ( r)-[c]verapamil and tariquidar . in conclusion , our results show that substantial inhibition of pgp can be safely achieved at the human bbb using an experimental ( nonmarketed ) pgp inhibitor , which results in pronounced increases in brain delivery of a radiolabelled pgp substrate .
this approach may be of interest to improve brain penetration of therapeutic drugs , which are cleared from brain by pgp .
moreover , we experimentally verified the hypothesis that pet analysis of baseline uptake ( i.e. , without administration of a pgp inhibitor ) of a radiolabelled pgp substrate in the pituitary gland gives an estimate of brain uptake when pgp is fully inhibited , thus providing the possibility to predict the maximum possible effect of pgp - mediated drug drug interactions at the bbb .
finally , our data provide much needed scaling factors for extrapolation of rodent data to humans in pharmacokinetic modelling .
| as p - glycoprotein ( pgp ) inhibition at the blood brain barrier ( bbb ) after administration of a single dose of tariquidar is transient , we performed positron emission tomography ( pet ) scans with the pgp substrate ( r)-[11c]verapamil in five healthy volunteers during continuous intravenous tariquidar infusion . total distribution volume ( vt ) of ( r)-[11c]verapamil in whole - brain gray matter increased by 27378% relative to baseline scans without tariquidar , which was higher than previously reported vt increases . during
tariquidar infusion whole - brain vt was comparable to vt in the pituitary gland , a region not protected by the bbb , which suggested that we were approaching complete pgp inhibition at the human bbb . |
despite its potential for use in organic synthesis , applications of tmsccl3 ( tms = sime3 ) have been far narrower in scope than those of closely related tmscf3 ( ruppert prakash reagent).1 two reasons can be identified as the origins of this situation .
firstly , all present literature preparations of tmsccl3 provide either low - to - modest isolated yields,2 or rely on extreme low - temperature protocols ( typically 110 c),2 limiting easy access to this reagent .
secondly , most applications of tmsccl3 require its activation by a silylphilic promoter , typically a fluoride ion .
the intimate mechanism(s ) by which this process proceeds are presently based on ad hoc suggestions rather than tangible data . in such environments it is possible to select reaction conditions that may not be mechanistically optimal .
although tmsccl3 is known to participate in a small number of 1,2-additions to aldehydes,3 ketones3 and imine derivatives ( scheme 1),4 1,4 addition modes are practically unknown and are limited to just six examples , with modest yields , in a single paper by cunico and zhang.5 we were interested to identify improved experimental conditions for such reactions and to understand the underlying mechanism of 1,4-addition of tmsccl3 .
new access to -ccl3-substituted nitroalkanes is of interest , and sosnovskikh et al . , and others,6 have developed a range of unusual and useful methods around this motif .
for the conjugate - addition mechanistic investigations we required access to large amounts of highly pure tmsccl3 . unfortunately , current literature preparations2 have significant limitations , either in yield , purity of reagent attained , reproducibility or the need for special conditions .
these issues are due to the fragility of mccl3 ( typically m = li or na ) intermediates .
0.1 per ml ) as a co - solvent ( 5 equiv ) would significantly enhance ccl3 capture , improving the tmsccl3 yield .
addition of lihdms ( lithium hexamethyl disilazide ) in hexane / thf ( 65:20 ) to a chloroform / tmscl ( 12:64 ) mixture at 65 c , followed by slow warming to ambient temperature proved optimal . after an appropriate workup ,
kugelrohr sublimation routinely afforded pristine material in 6470 % yield on a > 10 g scale .
the nitroolefins , 1 , for our study were prepared by using a one - pot procedure by dauzonne and royer,7 a condensation method by andrew and raphael8 or by using a very recent agno2 method by maiti et al.9 ( scheme 2 ) . the advantage of the former two methods , although the yields are often modest , is that they are technically simple and provide a direct route to the 3-nitro-2h - chromenes and styrenyl systems , respectively .
initial studies on substrate 1 a ( table 1 ) confirmed the findings of cunico and zhang,5 but indicated that csf is an unreliable promoter because of its low solubility in organic solvents .
soluble nbu4x [ x = cl , oac , f and especially ph3sif2 ( tbat ) ] were found to be efficient promoters at 5 mol % in both polar ( thf , entries 25 ) and non - polar ( toluene , entries 610 ) solvents .
the less - silylophilic promoter , nbu4br , led to a very slow turnover . due to the speed of the reactions in thf at 20 c no rate estimates could be attained ( entries 25 ) .
however , approximate initial rates ( based on conversion in the first 20 sec ) could be attained in toluene , confirming the high efficacy of tbat ; in the absence of any promoter no reaction occurred .
the spectroscopic properties of 2 a are in accord with 1,4-addition of the trichloromethyl group .
in particular , a characteristic multiplet is seen at h=5.56 ppm in the h nmr spectrum , correlating to the c nmr ch signal to the nitro group at c=80.7 ppm .
the -ch group is diagnostically shifted to lower frequency ( c=56.3 ppm in 2 a ) compared with its cch precursor ( c=139.2 ppm in 1 a ) , whereas a low - intensity quaternary signal at c=100.8 ppm is assigned to ccl3 and the molecular ion of 2 a shows the expected cl3 isotope pattern .
promoter comparison for tmsccl3 addition to 1 a. [ a ] carried out on 0.3 mmol 1 a ( 0.06 m ) .
yield data obtained from gc analysis in the presence of an internal standard ( 1-methylnaphthalene , 25 l , 0.18 mmol ) .
[ b ] use of either solvent resulted in inefficient catalysis ; [ c]>95 % of 2 a could only be attained after 24 h in thf with excess csf ( 3.75 equiv ) .
the conditions of table 1 ( entry 5 ) could be applied to a range of nitroalkene substrates , leading to various 1,4-addition products in 3795 % isolated yields ( scheme 3 ) . for the acyclic systems reversal of the addition mode
isolated addition products from tbat - catalysed ( 5 mol % ) additions of tmsccl3 to nitroalkenes 1 . the connectivity of 2 d could be confirmed by an x - ray crystallographic study ( figure 1 ) . in comparison ,
the 27 structures in the cambridge crystallographic database6 showing the same no2chchccl3 motif have : nc 1.491.53 , cc 1.521.55 and cccl3 1.511.57 ; n- c - c 105.8117.1 and c - c - ccl3 111.2117.1 .
two closely related six - ring structures ( cibgif and hacjay ) show anti arrangements , as in 2 d , but a syn motif is also known ( qemzue).6 molecular structure of 2 d. selected interatomic distances and angles : c(1)c(4 ) 1.556 , c(3)c(4 ) 1.544 , n(1)c(3 ) 1.519 ; c(1)-c(4)-c(3 ) 109.2 , n(1)-c(3)-c(4 ) 111.3 .
the following scope and limitation comments should be made : i ) addition of tmsccl3 to the substrate and tbat catalyst was appropriate for -substituted substrates 1 a e and 1 q. however , for terminal nitroalkenes ( 1 f p ) the alkene needed to be added slowly ( over 1 h ) to tmsccl3/tbat mixtures to avoid polymerisation , which led to unacceptable yields of 2 .
ii ) acyclic -substituted nitroalkenes led to very poor reactions and this substrate class was not further pursued ; -substitution provided clean products ( e.g. 2 m ) , but in reduced yield .
iii ) although alkyl , ether , ester , alkene and cf functional groups were tolerated in various degrees , some aryl bromide - containing substrates ( e.g. 8-bromo-3-nitro-2h - chromene ) and benzylic ( e)-bnchchno2 were not tolerated and led only to decomposition . given the known relative stability of the ccl3 radical , and its propensity to add to unsaturated systems ( kharasch et al.10 ) , tests were carried out to assess the veracity of such reaction pathways .
firstly , it was observed that the presence of the known radical inhibitors hydroquinone or butylated hydroxytoluene ( bht ) did not prevent tbat - catalysed additions to 1 d under strictly anaerobic conditions .
secondly , the substrate 1 r was used to provide potential intramolecular radical trapping sites .
the standard reaction conditions ( slow addition of 1 r to tmsccl3/tbat under argon ) led to trace amounts ( 15 % ) of bicyclic 2 r , and the majority of the starting material remained unconverted after the typical reaction time of 116 h ( scheme 4 ) .
however , if the reaction was conducted under aerobic conditions 2 r became the major product .
tmsccl3 solutions in thf , in the presence of tbat and o2 ( one molar equivalent of oxygen injected into a sealed reaction ) , were analysed by si nmr spectroscopy and revealed a significant amount of a single silicon - containing species showing si=7.4 ppm . based on comparison with literature silicon nmr shift values11 we assigned this new species as tms2o .
one explanation for the formation of 2 r is reaction of tmsccl3 ( in the presence of tbat ) with o2 , leading to tms2o2 and ccl3 radicals that cascade to 2 r , via 3 and 4 . although we could not detect any tms2o2 peroxide ( si27 ppm12 ) in air or in o2-exposed samples of tmsccl3 , in the presence or absence of tbat , the latter was rapidly converted to tms2o .
it is likely that any peroxide would be both generated and decomposed as shown in scheme 4 .
literature bicycles related to 2 r have been generated , either by oxidation of nitronate anions13 or through nitrile oxide formation and subsequent [ 2 + 3 ] cycloaddition chemistry,13 from the expected 1,4-addition product 5 .
although we can not completely exclude such possibilities , such approaches normally require stronger oxidants than molecular oxygen or prolonged heating at 60 c.13 the connectivity of 2 r could be confirmed by an x - ray crystallographic study on its dehydrochlorination product 2 r , obtained through simple mgso4 drying / recrystallisation of 2 r ( figure 2 ) .
formation of the cccl2 bond is also evident in the c nmr spectrum , in which the diagnostic ccl3 signal at c=99.4 ppm in 2 r is replaced by two quaternary alkene signals ( c=121.4 and 126.7 ppm ) .
both 2 r and 2 r have a cn resonance ( c=167.6 and 166.5 ppm , respectively ) . the structure of 2 r is reminiscent of other tetrahydro-3h - cyclopenta[c]isoxazoles in the cambridge crystallographic database.14 aerobic cyclisation of substrate 1 r. molecular structure of 2 r. selected interatomic distances and angles : n(1)c(6a ) 1.258 , n(1)o(2 ) 1.431 , c(6)c(7 ) 1.324 ; c(6a)-n(1)-o(2 ) 107.5 , c(7)-c(6)-c(6a ) 127.1 . despite the aerobic cyclisation shown in scheme 4
in particular , the observation of low , but reproducible , ee values in the formation of 2 q by using chiral promoters at low substrate conversion suggested that a different reaction mechanism operates under o2-free conditions .
to the best of our knowledge , no study of the explicit reaction mode of tmsccl3 with nbu4siph3f2 ( tbat ) has been carried out , therefore , we sought to define the non - radical process by multi - nuclear nmr studies .
reagent concentrations of 0.125 m in thf/[d6]benzene ( 5:1 ) at 20 c offered the best compromise with respect to solubility , si sensitivity and attainment of o2-free conditions .
temperatures of 20 c are also the lowest at which viable catalytic reactions are possible , indicating that the reactions should be slowed to only primary events at this temperature .
in an initial set of conditions at 20 c , tmsccl3 ( si=21.9 ppm ) was immediately converted to tmsf ( si=32.4 ppm)15 on addition of tbat ( si=108.8 ppm),16 which itself was transformed to ph3sif ( si=4.1 ppm)17 ( figure 3 ) .
no other silicon - containing species were present , except for traces of tms2o ( si=7.3 ppm)11 ( which could be minimised / eliminated by good experimental technique to eliminate the last traces of o2 ) .
the jsif coupling pattern is indicative of the number of attached fluorine atoms in these species .
the residual tbat species , tmsf and ph3sif could be correlated to signals at f=98.9,17 158.015 and 170.5 ppm16 in the f nmr spectrum of the reaction mixture at 20 c ( see the supporting information ) .
the si and f nmr spectra of the same reaction mixture at + 20 c show only very slight broadening , indicating that any exchange between the species detected is , at best , very slow under the reaction conditions . at 20
c the c nmr spectrum of the tmsccl3/tbat mixture , in the methyl region , confirmed the presence of me3sif ( c=0.4 ppm , jcf=15.5 hz ) and a singlet peak ( c=1.6 ppm ) ascribed to the expected exchange product , nbu4ccl3 . this latter compound is stable at 20 c indefinitely , no evidence of formation of tetrachlorethene ( c=120.7 ppm ) , or any other ccl3 or dichlorocarbene - derived byproducts was seen in the spectra .
no reaction was observed when nitroalkene 1 a was added last to the above mixture , which was then warmed from 20 to 0 c ( conditions under which the catalytic reaction is spontaneous ) .
si nmr spectra ( 79.5 mhz , 5:1 thf/[d6]benzene , 20 c ) : a ) tmsccl3 ( =21.9 ppm ) ; b ) tbat ( =108.8 ppm , j(sif)=254 hz ) ; c ) a nominal 1:1 mixture of tmsccl3/tbat ( a slight excess of tbat was used to provide a spectral internal standard ) , tmsf ( =32.4 ppm , j(sif)=275 hz ) , tms2o ( =7.3 ppm ) , ph3sif ( =4.1 ppm , j(sif)=282 hz ) . in a separate set of conditions , nitroalkene 1 a was first added to tmsccl3 at 20 c . the h nmr spectrum in the region h=6.88.0 ppm contains the aryl and alkene signals of 1 a ( see figure 4 a , b ) .
the signal of tmsccl3 is at h=0.20 ppm ( not shown in figure 4 ) .
the equivalent c nmr spectra confirm < 5 % reaction of alkene and tmsccl3 because only the characteristic peaks of 1 a and tmsccl3 , at c=4.5 ppm are present .
subsequent addition of tbat at 20 c to this mixture leads to partial ( 38 % ) , but immediate , conversion of 1 a into a new compound with h ( figure 4 c ) and c nmr spectra ( see the supporting information ) that are closely related to those of the addition product 2 a. this new species is assigned as nitronate 8 ( scheme 5 ) . in some experiments traces of a new species could be detected before the addition of tbat ( see figure 4 b ) .
this species could only be identified as a product of nitroalkene decomposition , or species 6 ( see below ) .
partial h nmr spectra ( 5:1 thf/[d6]benzene , 20 c ) : a ) alkene 1 a ; b ) a nominal 1:1 mixture of alkene 1 a and tmsccl3 ; c ) after addition of 1 equiv of tbat to mixture ( b ) .
signals due to tbat are marked ( ) , those due to ph3sif ( ) and those due to the proposed nitronate product 8 ( ) .
overall , the data from the stoichiometric nmr experiments are in accord with scheme 5 .
once formed , nbu4ccl3 is insufficiently nucleophilic to directly attack the nitroalkene and equilibration back to tmsccl3 is not possible .
rather , the nitroalkene binds tmsccl3 by means of an electron - rich no contact , providing 6
. we could detect no exchange broadening of the minor signals observed in figure 4 b from ambient temperature down to 20 c .
further cooling of the reaction was not possible because of the formation of non - homogeneous samples , and heating the sample was not possible .
however , the data are generally in accord with attack of an external nucleophile , in this case fluoride ions ( either directly or indirectly from tbat18 ) , on 6 , triggering ccl3 transfer , presumably through a chair - like transition state , leading to 7 . finally , due to the strength of the sif bond in tmsf ( and the clear lack of exchange with this species in the nmr studies )
if the conclusions of the stoichiometric reactions shown in scheme 5 can be translated to the catalytic reactions , three clear predictions can be made : i ) nitronate anions themselves should be excellent promoters of the reaction .
ii ) if the concentration of nitronate ( or indeed any other anionic promoter nucleophile ) builds up over time , a wide range of nucleophilic promoters ( nu ) will be available to replace fluoride ( f ) in the key conversion of 6 into 7 ( scheme 5 ) .
iii ) under such conditions , the ee value of the 1,4-addition product , produced by an asymmetric source of nu should decrease over time ( due to competition with an increasingly populated pool of promoter anions ) . to check these suppositions we prepared nbu4[ch2no2 ] from nitromethane and found that it does indeed promote rapid quantitative conversion of 1 a into 2 a.
the chiral complex [ ni(duphos)2](acac)2 ( duphos=(r , r)-methylduphos , cas [ 147253 - 67 - 6 ] ; acac = acetylacetonate ) was found to be an , albeit poor , promoter of asymmetric trichlomethylation of 1 q ( see the supporting information ) .
nevertheless , the ee value of 2 q formed by using this catalyst ( 10 mol % ) does decrease reproducibly from 15 to < 1 % over 20 h , in line with predictions .
the bifunctional chiral catalyst by takemoto et al.19 ( 10 mol % ) , associated with tbaf as a co - promoter ( 10 mol % ) , also led to a decrease in the ee value of 2 q over time ( from 26 % to < 1 % ) .
based on all of the data it seems likely that scheme 6 is the most rational description of the catalytic cycle .
the nitronate product , 8 , can either act as a promoter itself or leave the promoter pool by means of a reaction with either tmsx ( x = ccl3 or nu , if nu is a suitable leaving group ) . proposed catalytic cycle .
finally , the use of the 1,4-addition products , 2 , for the formation of other products was briefly investigated .
treatment of 2 a with kotbu in thf led to the formation of dichlorocyclopropane 9 in moderate yield ( scheme 7 ) , through -ch deprotonation .
for example , 2 f led to the formation of dichloroalkene 10 when treated under the same conditions .
catalytic 1,4-additions of tmsccl3 to electron - deficient michael acceptors have considerable potential for use in organic chemistry .
the mechanistic studies presented here are consistent with pre - coordination of the nitroalkene to the silicon reagent before its promotion by a silylphilic nucleophile .
attempts to develop asymmetric versions of this reaction will prove to be challenging for mechanistic reasons .
the kinetically derived nitronate product of the reaction is itself a highly effective chain carrier , and attaining a competitive chiral catalyst or alternative conditions will be critical for success .
full details of all transformations and associated spectroscopic data are given in the supporting information .
nitroolefins 1 a e were prepared by dauzonne and royer s one - pot procedure7 .
alkene 1 a showed identical spectroscopic properties to previously reported samples.20 compounds 1 b e , previously unreported , were fully characterised ( see the supporting information ) . nitroolefins 1 g
l were prepared by andrew and raphael s condensation method8 and had identical spectroscopic properties to previously reported samples.9 , 21 , 22 nitroolefins 1 m r were prepared by the agno2 method by maiti et al.,9 and had identical spectroscopic properties to previously reported samples.9 , 23 trimethyl(trichloromethyl)silane ( tmsccl3 ; 0.21 g , 1.1 mmol ) in thf ( 2 ml ) was added dropwise to a solution of the cyclic nitroalkene ( 1 mmol ) and tetrabutylammonium triphenyldifluorosilicate ( tbat ; 0.027 g , 5 mol % ) in thf ( 2 ml ) under argon at room temperature , and the reaction mixture was stirred overnight .
the mixture was concentrated in vacuo and then purified by flash chromatography on silica gel to give the corresponding michael addition products .
alternatively , the reactions were quenched with saturated nh4cl ( aq ) , extracted with ethyl acetate , dried over anhydrous mgso4 and concentrated before purification by chromatography .
the acyclic alkene ( 1 mmol ) in thf ( 2 ml ) was added dropwise , over a period of one hour , to a solution of trimethyl(trichloromethyl)silane ( tmsccl3 ; 0.21 g , 1.1 mmol ) and tetrabutylammonium triphenyldifluorosilicate ( tbat ; 0.027 g , 5 mol % ) in thf ( 2 ml ) under argon at room temperature , and the mixture was stirred overnight .
the mixture was concentrated in vacuo and then purified by flash chromatography on silica gel to give the corresponding michael addition products .
trimethyl(trichloromethyl)silane ( tmsccl3 ; 0.21 g , 1.1 mmol ) in thf ( 2 ml ) was added dropwise to a solution of the cyclic nitroalkene ( 1 mmol ) and tetrabutylammonium triphenyldifluorosilicate ( tbat ; 0.027 g , 5 mol % ) in thf ( 2 ml ) under argon at room temperature , and the reaction mixture was stirred overnight .
the mixture was concentrated in vacuo and then purified by flash chromatography on silica gel to give the corresponding michael addition products .
alternatively , the reactions were quenched with saturated nh4cl ( aq ) , extracted with ethyl acetate , dried over anhydrous mgso4 and concentrated before purification by chromatography .
the acyclic alkene ( 1 mmol ) in thf ( 2 ml ) was added dropwise , over a period of one hour , to a solution of trimethyl(trichloromethyl)silane ( tmsccl3 ; 0.21 g , 1.1 mmol ) and tetrabutylammonium triphenyldifluorosilicate ( tbat ; 0.027 g , 5 mol % ) in thf ( 2 ml ) under argon at room temperature , and the mixture was stirred overnight .
the mixture was concentrated in vacuo and then purified by flash chromatography on silica gel to give the corresponding michael addition products .
as a service to our authors and readers , this journal provides supporting information supplied by the authors .
such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors | improved synthetic conditions allow preparation of tmsccl3 in good yield ( 70 % ) and excellent purity .
compounds of the type nbu4x [ x = ph3sif2 ( tbat ) , f ( tetrabutylammonium fluoride , tbaf ) , oac , cl and br ] act as catalytic promoters for 1,4-additions to a range of cyclic and acyclic nitroalkenes , in thf at 025 c , typically in moderate to excellent yields ( 3795 % ) .
tbat is the most effective promoter and bromide the least effective .
multinuclear nmr studies ( 1h , 19f , 13c and 29si ) under anaerobic conditions indicate that addition of tmsccl3 to tbat ( both 0.13 m ) at 20 c , in the absence of nitroalkene , leads immediately to mixtures of me3sif , ph3sif and nbu4ccl3 .
the latter is stable to at least 0 c and does not add nitroalkene from 20 to 0 c , even after extended periods .
nitroalkene , in the presence of tmsccl3 ( both 0.13 m at 20 c ) , when treated with tbat , leads to immediate formation of the 1,4-addition product , suggesting the reaction proceeds via a transient [ me3si(alkene)ccl3 ] species , in which ( alkene ) indicates an sio coordinated nitroalkene . the anaerobic catalytic chain is propagated through the kinetic nitronate anion resulting from 1,4 ccl3 addition to the nitroalkene .
this is demonstrated by the fact that isolated nbu4[ch2no2 ] is an efficient promoter .
use of h2cch(ch2)2chchno2 in air affords radical - derived bicyclic products arising from aerobic oxidation . |
the ratio of copper ( cu ) to other dietary components ( e.g. , zinc ( zn ) , iron , sulfate , and molybdenum ) may be as important as the actual cu levels in the diet .
cu / zn ratios may be important to the adequate metabolism of cholesterol , with low ratios resulting in hypercholesterolemia .
previous studies have focused on acute severe cu deficiency , which is relatively rare in humans and animals on typical varied diets .
the determination of cu needs and marginal deficiency is complicated by the fact that while cu deficiency does not necessarily lower the level of cu - dependent enzymes , it does significantly lowers their activity .
superoxide dismutase ( sod ) functions as an antioxidant by catalyzing the conversion of superoxide radicals ( free radicals or reactive oxygen species ( ros ) ) to hydrogenperoxide , which can subsequently be reduced to water by other antioxidant enzymes .
superoxide radicals may react with other ros such as nitricoxide to form highly toxic species like peroxynitrite , in addition to its direct toxic effects .
peroxy nitrite reacts with the tyrosine residues in proteins resulting with the nitrotyrosine production in plasma proteins , which is considered as an in direct evidence of peroxynitrite production and increased oxidative stress .
although nitrotyrosine was not detectable in the plasma of the healthy controls , nitrotyrosine was found in the plasma of all type 2 diabetic patients ( type 2 diabetes mellitus ( t2 dm ) ) examined .
previous studies correlated plasma nitrotyrosine values with plasma glucose concentrations and found a significant positive correlation .
furthermore , exposure of endothelial cells to high glucose level leads to an augmented production of superoxide anion , which may quench nitric oxide level resulting in impaired endothelial functions , vasodilation , and delayed cell replication .
alternatively , superoxide can be dismutated to a much more reactive hydrogen peroxide , which through the fenton reaction can then lead to a highly toxic hydroxyl radical formation .
two forms of sod contain cu : i ) cu / zn - sod is found within most cells of the body , including red blood cells , and ii ) extracellular ( ec)-sod is a cu - containing enzyme found in high levels in the lungs and low levels in blood plasma . almost all of the cu in our bodies is bound either to transport proteins ( ceruloplasmin and cu - albumin ) , storage proteins ( metallothioneins ) , or cu containing enzymes .
intracellular metallothionine normally stores little cu providing protection from the harmful effects of free cu .
ceruloplasmin may function as an antioxidant in two different ways : by binding to cu , ceruloplasmin prevents free cu ions from catalyzing oxidative damage .
the other way is through the oxidation of ferrous iron by ceruplasmin , facilitating iron load into its transport protein , transferrin , and preventing free ferrous ions from participating in harmful free radical generating reactions .
major reason for the decreased sod activity is the glycosylation of cu / zn - sod which has been shown to lead to enzyme inactivation both in vivo and in vitro .
also cu / zn - sod cleavage and release of cu
in vitro resulted intransition metalcatalyzed ros formation .
erythrocyte cu / zn - sod activity correlated inversely with indices of glycemic control in dm patients .
however , red cell cu / zn - sod activity has also been found to be decreased in dm patients .
glycation may decrease cell - associated ec - sod , which could predispose to oxidative damage .
earlier reports found decreased red cell cu / zn - sod activity in dm patients with retinopathy compared to dm patients without microvascular complications and nondiabetic control subjects .
this study was approved by the scientific and ethics committee of the college of medicine , al - nahrain university .
ninety - two participants were recruited for this study ( 55 participants with t2 dm and 37 normal control subjects ) .
type 2 diabetic patients ( n = 55 ) were divided according to the urine protein ( albumin ) excretion measured in g / mg creatinine [ table 1 ] into : demographic and clinical data of the participants included in the study
patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31).patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) .
patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31 ) .
patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) .
all patients were recruited from the outpatient diabetes clinic at al - kadhymia teaching hospital .
the exclusion criteria included : patients with any recent medical illness ; impaired thyroid or renal function ; diagnosis of renal disease ; and treatment with estrogen , glucocorticoids , or other drugs except oral hypoglycemic and/or beta blocker antihypertensive drugs .
all patients included in the study were nonsmokers ; none were taking antioxidant supplements or drugs with known antioxidant activity .
the control group consisted of 37 healthy , age- and gender - matched subjects ( 48.92 8.9 years ) .
the control group consisted of participants with no known medical history and with no family history of diabetes or nephropathy .
a total of 10 ml of venous blood samples were collected from each subject in the study after 10 - 12 h fasting .
two milliliters were collected into ethylene diaminetetraa cetic acid ( edta ) containing tubes for glycated hemoglobin ( hba1c ) assay .
the remaining 8 ml were centrifuged at 3,000 rpm for 10 min after about 30 min from the time of blood collection .
all assays were obtained by running duplicates for the test , control , and the standard .
random morning urine specimens were obtained from each subject in the study , to quantify albuminuria , creatinine , cu , and albumin to creatinine ratio .
no urine preservatives were used ; the samples were stored in appropriate containers and were kept at the refrigerator until the time of measurements .
a. methods applied in urine : a micro method was employed for the determination of urinary protein based upon the coprecipitation of protein and ponceau s dye by trichloracetic acid ( tca ) , dissolution of the precipitate in dilute alkali , and spectrophotometric determination of the dye in alkaline solution .
urinary creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
b. methods applied in blood : serum creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
a stock standard concentration of cu ( 50 mol / l ) was prepared and subsequent dilutions were made to obtain a calibration curve .
urine samples were diluted ( 1:10 ) by deionized water and measured directly against an aqueous standard made from certified standard solution .
cu hallow cathode lamps were used at wavelength of 324.75 nm . these solutions were aspirated directly into air - acetylene flame .
serum lipids were measure using biomerieux assay kits for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) .
serum sod was measured using the sod assay kit - water soluble tetrazolium salt ( dojindo molecular technologies , rockville , md , usa ) .
glycated hemoglobin ( hba1c ) samples were by varianttm hba1c program , which is intended for the determination of hba1c in human whole blood using ion - exchange high performance liquid chromatography ( hplc ) .
statistical significance was determined by anova test followed by unpaired student 's t - test and pearson 's correlation ( r ) to test correlation of regression .
this study was approved by the scientific and ethics committee of the college of medicine , al - nahrain university .
ninety - two participants were recruited for this study ( 55 participants with t2 dm and 37 normal control subjects ) .
type 2 diabetic patients ( n = 55 ) were divided according to the urine protein ( albumin ) excretion measured in g / mg creatinine [ table 1 ] into : demographic and clinical data of the participants included in the study
patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31).patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) .
patients with albumin - creatinine ratio that is equal to 30 - 299 g / mg were considered to have microal buminuria ( n = 31 ) .
patients with albuminexcretion less than 30 g / mg creatinine were considered normoal buminuric ( n = 24 ) .
all patients were recruited from the outpatient diabetes clinic at al - kadhymia teaching hospital .
the exclusion criteria included : patients with any recent medical illness ; impaired thyroid or renal function ; diagnosis of renal disease ; and treatment with estrogen , glucocorticoids , or other drugs except oral hypoglycemic and/or beta blocker antihypertensive drugs .
all patients included in the study were nonsmokers ; none were taking antioxidant supplements or drugs with known antioxidant activity .
the control group consisted of 37 healthy , age- and gender - matched subjects ( 48.92 8.9 years ) .
the control group consisted of participants with no known medical history and with no family history of diabetes or nephropathy .
a total of 10 ml of venous blood samples were collected from each subject in the study after 10 - 12 h fasting .
two milliliters were collected into ethylene diaminetetraa cetic acid ( edta ) containing tubes for glycated hemoglobin ( hba1c ) assay .
the remaining 8 ml were centrifuged at 3,000 rpm for 10 min after about 30 min from the time of blood collection .
all assays were obtained by running duplicates for the test , control , and the standard .
random morning urine specimens were obtained from each subject in the study , to quantify albuminuria , creatinine , cu , and albumin to creatinine ratio .
no urine preservatives were used ; the samples were stored in appropriate containers and were kept at the refrigerator until the time of measurements .
a. methods applied in urine : a micro method was employed for the determination of urinary protein based upon the coprecipitation of protein and ponceau s dye by trichloracetic acid ( tca ) , dissolution of the precipitate in dilute alkali , and spectrophotometric determination of the dye in alkaline solution .
urinary creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
b. methods applied in blood : serum creatinine was estimated by the biomerieux assay kit based on the method of bartels et al .
a stock standard concentration of cu ( 50 mol / l ) was prepared and subsequent dilutions were made to obtain a calibration curve .
urine samples were diluted ( 1:10 ) by deionized water and measured directly against an aqueous standard made from certified standard solution .
cu hallow cathode lamps were used at wavelength of 324.75 nm . these solutions were aspirated directly into air - acetylene flame .
serum lipids were measure using biomerieux assay kits for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) .
serum sod was measured using the sod assay kit - water soluble tetrazolium salt ( dojindo molecular technologies , rockville , md , usa ) .
glycated hemoglobin ( hba1c ) samples were by varianttm hba1c program , which is intended for the determination of hba1c in human whole blood using ion - exchange high performance liquid chromatography ( hplc ) .
statistical significance was determined by anova test followed by unpaired student 's t - test and pearson 's correlation ( r ) to test correlation of regression .
all groups were closely age - matched , and the two diabetes groups were well - matched for duration of disease [ table 1 ] .
microabuminuric diabetic group shows a significant increase in the mean urinary cu / creatinine ratio when compared with controls ( p < 0.05 ) , while normoal buminuric diabetic patients shows an insignificant increase , p > 0.05 in mean urine cu / creatinine ratio when compared with controls [ table 2 ] .
urinary copper excretion and serum superoxide dismutase enzyme levels in microal buminurics , normoal buminurics , and control subjects serum sod was significantly decreased in the diabetes microal buminuric group compared to the control group ( p < 0.05 ) , such a significant correlation was not seen with the diabetes normoal buminuric group [ figure 1 and table 2 ] .
studies showed a reduction in erythrocyte sod and catalase ( cat ) activities in subjects with impaired glucose tolerance ( igt ) , early hyperglycemia , and type 2 dm patients .
however , other studies showed that the activities of these enzymes were within normal range in t2 dm patients in poor glycemic control .
ec - sod activity was found to be similar in t1 dm patients despite some what higher plasma ec - sod levels .
red cell cu / zn - sod activity was similar in t1 dm and t2 dm patients compared to normal subjects , irrespective of microvascular complications .
leukocyte sod activity was similar between type 2 dm patients and healthy control subjects , despite increased lipidperoxidation and decreased ascorbate levels . while the role of adequate cu levels in maintaining cardiovascular health is well - established , there are still inconsistent data about the correlation of cu with persisting hyperglycemia .
some studies showed an elevation of serum cu , while other studies showed a significant reduction of cu in diabetes .
many researchers have considered this elevation of serum cu to play a role in the pathogenesis of cardiovascular disease , although other researchers have strongly disagreed with this hypothesis .
an animal study , however , seems to have explained this relationship between cu levels and cardiovascular disease .
this study examined the effects of diet - induced atherosclerosis on the cu levels and status of numerous tissues .
it was found that serum cu levels increase significantly , while aorta and liver cu levels decrease significantly , in rats with experimental atherosclerosis .
so instead of assuming that these elevated cu levels contribute to the formation of atherosclerosis , these researchers examined the effects of increasing the dietary cu levels in these animals .
administration of additional cu resulted in a further increase in serum cu , a significant decrease in serum cholesterol , and an increase and normalization in aorta and liver cu levels .
however , instead of increasing the incidence of atherosclerosis , additional cu significantly decreased the incidence of atherosclerosis in the aorta and coronary arteries .
further , it has been shown that excess dietary cholesterol causes cardiovascular disease by lowering the absorption of cu , an effect that is preventable by increasing the cu level in the diet .
however , increased urinary albumin loss has been postulated to be a marker of a generalized increase in vascular permeability , which might predispose to greater penetration into the arterial wall of atherogenic lipoprotein particles .
urinary cu concentrations significantly increased only in microal buminuric patients , similar results were found by , but with macroal buminuric diabetic patients . in diabetic patients with advanced nephropathy
, urinary cu excretion may be due to dissociations from both cu - albumin and ceruloplasmin - cu complexes filtered through the damaged glomerulus .
overloading of urinary cu to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy .
therefore , regulation of the blood concentrations of cu may be a potential therapeutic target for prevention and treatment of diabetic nephropathy .
however , there is still a lot to know about the mechanism of cu homeostasis at the cellular level .
if reduction of serum cu can be shown to have a protective effect against oxidative stress in dm , this may have a direct impact on the use of cu chelators as a safe therapeutic modality in diabetes . | background : copper ( cu ) is essential both for its role in antioxidant enzymes , like cu / zinc ( zn ) superoxide dismutase ( sod ) and ceruloplasmin , as well as its role in lysyl oxidase , essential for the strength and integrity of the heart and blood vessels . with such a central role in cardiovascular health , cu has been generally overlooked in the debate over improving our cardiovascular health .
cu deficiency has produced many of the same abnormalities present in cardiovascular disease .
it seems almost certain that cu plays a large role in the development of this killer disease , not because of its excess in the diet , but rather its deficiency.aim:this study was undertaken to investigate the cardiovascular effects of cu deficiency on the activity of sod in patients with type 2 diabetes mellitus ( t2 dm ) with and without diabetic nephropathy.materials and methods : fifty - five patients with t2 dm were recruited in this study which were divided into two subgroups based on the presence of microalbuminuria , the first group ( microal buminuric group , n = 31 ) had a microalbuminuria between 30 and 299 g / mg .
the second group ( normoal buminuric group , n = 29 ) had an albumin level less than 30 g / mg .
the two diabetic groups were compared to the control group ( n = 37).results : the results of our study showed a significant reduction in the levels of sod enzyme associated with an increased urinary cu excretion in microalbuminuric group compared to the control group at p <
0.05.conclusions:the current study illustrates that the regulation of the blood concentrations of cu may be a potential therapeutic target for prevention and treatment of diabetic nephropathy . |
breast milk , the sole source of nourishment
for newborns , has been
under intense selective pressure over millions of years of evolution
to meet the infant s needs to grow and survive .
interestingly , the third most abundant chemical component class
in human milk after lactose and lipids , human milk oligosaccharides
( hmos ) , is both non - nutritive and non - digestible .
hmos play a key role in creating and maintaining a healthy
infant gut microbiota through two established mechanisms .
first , hmos
have prebiotic effects ( promoting growth of beneficial bacteria such
as bifidobacterium and lactobacillus ) .
recent
metagenomic studies have confirmed that bifidobacteria are enriched
in the intestines of healthy breast fed infants . in vitro studies
provide a reason for the prevalence of
bifidobacterial and even bacteroides species : they selectively consume
hmos .
a number of studies have characterized the
enzymes in bifidobacteria that specifically degrade hmos ( reviewed
in ref ( 8) ) .
second , hmos compete for specific pathogen binding with sites in
the infant gut .
free hmos and conjugated
cell surface glycoforms are synthesized by similar glycosyltransferases
and thus have common epitopes .
ingested hmos can thus interact with
enterocyte surface molecules , limiting binding to these sites by viruses
such as hiv and rotavirus .
in addition , because hmos are also unbound ,
they can serve as free analogues of pathogen host receptors . instead
of binding to cell surface glycoproteins or glycolipids , pathogens
bind to hmos .
anti - adhesive activity
of free hmos has been described for vibrio cholerae , salmonella fyris , enteropathogenic and enterotoxigenicescherichia coli , streptococcus pneumoniae , and caliciviruses .
for instance , fucosylated
hmos inhibit the binding of campylobactor jejuni to intestinal cells , whereas sialylated
hmos block the adhesion of e. coli to
human erythrocytes .
the functions
of hmos are related to their specific structures ;
however , identifying and quantifying those structures has been a major
challenge in hmo analysis .
hmos can have diverse and complicated structures
even though they commonly share a lactose core consisting of glucose
and galactose linked via a -1,4-linkage .
functional studies to date have involved either a limited
number of structures or little or no structural information .
the benefits of breast feeding include the long - term health implications
of a well - established , specific , and protective microbiota .
recent
advances in glycomic analysis and genomic sequencing have allowed
us to observe the correlation between hmo consumption and gut microbiota
populations . in this study , we demonstrate two examples of direct
correlation between the abundances of hmos in feces and the composition
of the infant fecal microbiota as proof - of - concept .
hmos from fecal
samples of breast - fed infants were analyzed using advanced separation
and mass spectrometry methods that allow quantitation of specific
structures .
microbial populations were characterized by next - generation
sequencing of 16s rdna amplicons , specific qpcr , and bifidobacterium species - specific terminal restriction
fragment length polymorphism ( bif - trflp ) .
the observed shift in the
gut microbiota of these two healthy infants from a non - hmo - consuming
population into a more saccharolytic microbiota with a corresponding
decrease in fecal hmo intensities supports more extensive analyses
of larger populations .
the study was approved
by the institutional review board at uc davis ( protocol no . 200715509 - 4 ) ,
and informed consent was obtained from the parents of infants prior
to participation .
the infants included were born vaginally at term
and were not treated with antibiotics during the study period .
infant b had formula supplementation
for 4 days right after birth , from days 2 to 6 , and then was solely
breast fed .
details of the mothers diet and health status
were not collected as part of this study .
feces samples were
collected from two full - term infants as follows : twice a week for
the first month , twice a month in the second month , and once or twice
a month thereafter .
crude hmos were isolated
and purified from extracted feces through
a series of liquid- and solid - phase extractions , as described previously .
briefly , four volumes of chloroform / methanol ( 2:1 v / v ) were added
to the decanted liquid , and the mixture was centrifuged at 4000 g for 30 min at 4 c .
two volumes of ethanol were added , and the mixture was
left at 4 c overnight and then centrifuged for 30 min at 4 c .
the supernatant solution was evaporated to dryness using a centrifugal
evaporator ( savant aes 2010 ) .
hmos were reduced to alditol form
by adding sodium borohydride and incubating at 65 c for 1 h ,
purified by solid - phase extraction using c8 and graphitized carbon
columns , and evaporated to dryness .
oligosaccharides were profiled using matrix - assisted laser desorption / ionization
fourier transform ion cyclotron resonance mass spectrometry ( maldi
ft - icr ms , agilent , formerly varian , palo alto , ca , equipped with
an external promaldi source ) and nano - high - performance liquid chip / time - of - flight
( nano - hplc - chip / tof , agilent 6200 ) ms , as described previously . for maldi
ft - icr ms experiments , 2,5-dihydroxy - benzoic acid ( dhb )
was used as matrix ( 5 mg/100 l in 50:50 acn / h2o )
and sodium chloride ( 0.01 m in 50:50 acn / h2o ) was used
as cation dopant .
the precursor ions were detected primarily as [ m
+ na ] ions in the positive ionization mode .
identification
of hmo compositions was done based on accurate masses using glycan
finder software ( written in - house in igor pro 5.04b , wavemetrics ) ,
which filters experimental accurate masses to a specified mass tolerance
( set at 20 ppm ) based on accurate masses of theoretical oligosaccharides .
the nano - hlpc - chip / tof ms experiments , as described previously , were performed using a porous graphitized carbon hplc microchip
with a 40 nl enrichment column and 43 0.075 mm i.d./5 m
pore size analytical column .
separation was achieved using a binary
gradient solvent system with a ( 3% acn in 0.1% formic acid solution )
and b ( 90% acn in 0.1% formic acid solution ) .
the chip column was
equilibrated and eluted at a flow rate of 0.4 l for nanopump
and 4 l for capillary pump .
the gradient ran for 65 min and
was programmed as follows : 2.520 min , 016% b ; 2030
min , 1644% b ; 3035 min , b increased to 100% ; 3545
min , continue at 100% b ; and 4565 min , 0% b to allow equilibration
of the column prior to the next sample injection .
data were acquired
in the mass range of m / z 2003000
using electrospray ionization ( esi ) in the positive ionization mode .
deconvoluted peaks were extracted using the molecular feature of the
agilent mass hunter software at 20 ppm error .
ms searcher software ( in - house , written in java ) , which assigns structures
to a peak based on retention time and accurate mass .
lc ms
searcher uses a library of 45 neutral hmos and 30 acidic hmos characterized
using exoglycosidases and ms / ms by wu et al .
around 2000 spectra are inputted in the program per run , and with
the hmo library , the lc
ms searcher software identifies hmos
from all of the scans based on retention times and m / z values .
it then outputs intensities , structures ,
and deuterium / hydrogen ( d / h ) ratios of said hmos .
this library approach
allows for separation of isomeric species and identification of individual
structures based primarily on accurate masses and reproducible retention
times , since extensive confirmation of hmos by ms / ms and exoglycosidases
were done previously .
deuterium - labeled internal standards were
used for the relative quantification of hmos using maldi ft - icr ms ,
as described previously .
this method
gives a relative standard deviation of less than 15% and allows for
a more linear quantitation over a larger dynamic range .
briefly , pooled hmos isolated from milk of several
donor mothers were reduced using sodium borodeuteride , purified by
graphitized carbon solid - phase extraction , and used as reference solution .
both the deuterated reference solution and sample solution were spotted
as one sample onto the maldi plate and analyzed by maldi ft - icr ms
in the positive mode ( n = 3 ) .
the ratio of sample
reduced by nabh4 and standard reduced by nabd4 ( h / d ratio ) was calculated using microsoft excel using the formula
described below .
h / d ratios were used for quantitation as described
by ninonuevo et al.where m and n are the experimental intensities of a ( monoisotopic
peak ) and a + 1 , respectively , and p and q are calculated intensities of a and a + 1 , respectively . to compute the temporal
changes in the h to d ratios across a set
of samples ,
a reference sample is set and is used to compare with
all the other samples to quantitate oligosaccharide to the isomer level , both the
deuterated
reference solution and sample solution were also injected into the
nano - hplc - chip / tof ms .
ms
searcher ( the program outputs d / h ratios ;
we took the reciprocal ) and verified manually using microsoft excel .
bacterial genomic dna was
extracted from fecal samples in order to analyze the infant gut microbial
populations as previously described with a few modifications .
briefly , 200 mg of stool was resuspended 1:10
in ice - cold phosphate buffer solution ( pbs ) and centrifuged ( 8000 g for 5 min at room temperature ) ; then , the supernatant
was decanted .
this rinse was repeated twice , and the fecal pellet
was resuspended in 200 l of lysis buffer ( 2 mm edta , 1.2% tritonx-100 ,
20 mm tris - hcl , ph 8.0 ) with freshly added 40 mg / ml lysozyme .
buffer asl from qiaamp
dna stool mini kit ( qiagen , valencia , ca ) was added to equal 2.0 ml ,
and the sample was then vortexed until it was thoroughly mixed .
samples
were then homogenized by bead - beating in a fastprep-24 instrument
( mp biomedicals , solon , oh ) for 2 min at 6.5 m / s .
homogenate was incubated
for 5 min at 95 c , vortexed , and centrifuged at 13 000 g for 1 min to pellet stool particles .
1.2 ml of the supernatant
was used to purify dna with the qiagen stool mini kit according to
the manufacturer s instructions .
preparation of fecal dna samples for
pyrosequencing was carried out as previously described by the core for applied genomics and ecology
( cage , university of nebraska ) .
the v1v3 region of the 16s
rrna gene was pcr amplified using bar - coded universal primers with
roche-454 a or b titanium adapter sequences , shown in italics , b-8f
( 5-cctatcccctgtgtgccttggcagtctcagagagtttgatcmtggctcag-3 ) and a-518r ( 5-ccatctcatccctgcgtgtctccgactcagnnnnnnnnattaccgcggctgctgg-3 ) , where
the n represents the 8 base barcode sequence unique to each sample .
to ensure the representation of bifidobacteria
, bifidobacteria - specific primer b-8fbif
( 5-cctatcccctgtgtgccttggcagtctcagagggttcgattctggctcag-3 ) was mixed with
the 8f universal primer , which has a 3 base pair mismatch , at a concentration
of 4:1 . the pcr mixture and
sequencing was performed using
the 454 roche sequencing primer kit from the a end and carried out
using the standard protocol on a roche genome sequencer gs - flx .
the v4 region of the 16s rrna gene was pcr amplified using universal
barcoded primers with illumina sequencing adapters , shown in italics ,
the n represents the 8 bp barcode sequence unique to each sample ,
and the linker is in bold , v4f ( 5-aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatctnnnnnnnngtgtgccagcmgccgcggtaa-3 )
and v4rev ( 5-caagcagaagacggcatacgagatcggtctcggcattcctgctgaaccgctcttccgatctccggactachvgggtwtctaat-3 ) .
pcr reactions contained 12.5 l of 2 gotaq green master
mix ( promega , madison , wi ) , 1.0 l of 25 mm mgcl2 , 8.5 l of water , 0.5 l of forward and reverse primers
( 10 m final concentration ) , and 2.0 l of genomic dna .
amplicons were
combined and cleaned using the qiaquick 96 pcr purification kit ( qiagen ,
valencia , ca ) .
amplicon dna concentrations were quantified using the
quant - it picogreen dsdna kit in 96-well microplates , and fluorescence
detection , composite sample mixture , and gel purification were carried
out as previously described .
the sample
was sent to the university of california dna technologies core facility
for sequencing on an illumina genome analyzer ii sequencing platform .
the qiime software package ( version
1.4.0 ) was used to analyze the results of both the pyrosequencing
and illumina sequencing runs .
v1v3
16s rrna gene sequences from pyrosequencing reads were removed from
analysis if they were < 200 bp in length , contained > 3 ambiguous
bases , had a mean quality score < 25 , contained a homopolymer run
greater than 6 nt , or did not contain a primer or a barcode sequence .
similar sequences were clustered into operational taxonomic units
( otus ) with uclust software and minimum
identity of 97% .
taxonomy was assigned to each otu with the ribosomal database
project ( rdp ) classifier , with a minimum
support threshold of 80% and the rdp taxonomic nomenclature .
otu representatives
were aligned against the greengenes core set with pynast software with a minimum
alignment length of 150 bp and a minimum identity of 75% .
illumina v4 16s rrna gene sequences were demultiplexed and
quality - filtered using the qiime software package as well .
reads were truncated after a maximum number
of 3 consecutive low - quality scores .
after quality trimming , reads
were removed from analysis if they were < 60 bp and the number of
ambiguous bases was > 3 .
otu clustering , taxonomy assignment , phylogenetic
tree - building , and alignment were carried out as stated above , with
the exception that the minimum alignment length was 75 bp .
otus with
sequences that numbered less than 0.01% of the total reads were filtered
out to reduce noise in the assignments .
beta diversity
was estimated by computing weighted and unweighted unifrac distances
between samples .
pearson product moment correlation coefficients
were calculated in qiime between the
nano - hplc - chip / tof ms d / h ratios of individual hmo isomers normalized
to baseline intensity and the relative abundances of order - level bacterial
taxa .
sybr green and taqman qpcr assays were performed on
a 7500 fast real - time
pcr system ( applied biosystems , carlsbad , ca ) with primers specific
for universal bacteria , bacteroidales , and bifidobacterium , and species - specific bifidobacterium primers for b. longum group , b. adolescentis group , b. catenulatum group , b. breve , and b. bifidum .
sybr green assays
contained 10 l of 2 takara perfect real time master mix ,
6 l of water , 1 l each of forward and reverse primers ,
and 2 l of genomic dna .
bifidobacterium taqman qpcr assays contained 12.5
l of 2 taqman universal pcr master mix ( applied biosystems ) ,
2.5 l each of forward and reverse primers , taqman probe , 3.75
l of water , and 2.5 l of diluted genomic dna .
b. longum group taqman assays contained 10 l
2 taqman universal pcr master mix ( applied biosystems ) , 1 l
each forward and reverse primers and taqman probe , 5 l water ,
and 2 l genomic dna .
reaction conditions and primer concentrations
were as described in supporting information table
1 .
bifidobacterium genus - specific primers ( nbif389 , 5-gccttcgggttgtaaac ;
and nbif1018r , 5-gaccatgcacccctgtg-3 )
were designed targeting a consensus alignment of 16s rrna genes from
all bifidobacterium sequences deposited
in the ribosomal database project ( rdp ) database aligned using clustalx .
primer specificity
and taxonomic coverage were determined using primerprospector , checking against a representative subset of
the greengenes 16s rrna database filtered at 97% identity .
samples were amplified by pcr in 50 l
reactions containing 5100 ng of dna template , 25 l
of 2 promega gotaq green master mix ( promega , madison , wi ) ,
1 mm of mgcl2 , and 2 pmol of each primer .
each pcr was
performed in triplicate , and the products combined prior to purification .
the pcr conditions consisted of an initial denaturation at 95 c
for 2 min , followed by 30 cycles of denaturation at 95 c for
1 min , annealing at 51 c for 1 min , and extension at 72 c
for 2 min , with a final extension at 72 c for 5 min .
amplicons
were digested using alui and haeiii following the manufacturers
instructions for each enzyme .
the digested dna was submitted to the
uc davis college of biological sciences sequencing facility for fragment
analysis .
traces were visualized using the program peak scanner v1.0
( applied biosystems ) using a baseline detection value of 10 fluorescence
units .
peak filtration and clustering were performed with r software
using the ibest script suite .
otu picking
was based on an in silico digest database generated by the virtual
digest tool from mica of good - quality
16s rrna gene sequences compiled by the ribosomal database project
release 10 , allowing up to 3 nucleotide mismatches
within 15 bp of the 5 terminus of the forward primer .
fecal glycoprofiles and
microbial profiles were obtained for weeks 0 , 1 , 2 , and 13 .
previous
results have shown that the glycan composition is relatively constant
for mature milk over a 6 month period .
the amount of hmo ingested increases as milk consumption increases
over the first several weeks of life .
figure 1 shows the fecal oligosaccharide compositions ( analyzed by maldi
ft - icr ms ) and bacterial populations ( analyzed by 16s rdna pyrosequencing )
of infant a at the four time points .
corresponding
monoisotopic masses and compositions of the nominal masses in figure 1 are shown in table 1 .
the
table also shows monoisotopic masses of [ m + na ] ion ,
the commonly observed ion in maldi ft - icr ms .
exact masses and elemental
compositions were calculated using the nist glyco mass calculator
( de leoz and stein , http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:glycocalc ) .
changes in oligosaccharide intensities and bacterial population
in the feces of infant a. ( a ) percent change in h / d ratios of hmos
in the feces of infant a at weeks 0 , 1 , 2 , and 13 .
( b ) corresponding fecal bacterial population of infant
a using 16s rdna pyrosequencing .
the monoisotopic masses of the
[ m + na ] precursor ion , the commonly observed ion in maldi
ft - icr ms , are also shown .
hex , hexose ; hexnac , n - acetylhexosamine ; fuc , fucose ; neuac , n - acetylneuraminic
acid .
the graph in week 0 ( figure 1a )
should not be interpreted as each composition
having the same abundances .
each composition is composed
of several isomers , as discussed in greater detail below .
infant a
shows a general increase of all fecal hmos from week 0 to week 1 .
at week 2 , the amount of fecal hmos decreases to nearly the original
level of week 0 . at week 13 , the fecal hmos decrease to a fraction
of the peak amount at week 1 .
the decrease is not uniform ; there are
a number of compositions that increase or are unchanged at week 13 .
order - level fecal populations are shown in figure 2a , and genus level , in figure 2b .
multiplexed
pyrosequencing and illumina sequencing of infant a s samples
yielded 12 779 and 288 390 partial 16s rrna gene sequence
reads ( mean length 492 and 143 bp ) , respectively .
the mean number
of reads per sample was 5458 for pyrosequencing and 72 098
for illumina .
the observed early predominance of streptococcus in the gut ( figure 1b ) is consistent with previous observations in breast fed
infants .
previously demonstrated that tested species of streptococci
consumed hmo poorly or not at all . during the first week the fecal
oligosaccharides increase ; we hypothesize that they are being provided
by the mother but are not being consumed by the gut microbes .
the
amount of oligosaccharides in feces peaks during week 1 ; however ,
by week 2 , there is a noticeable decrease in intensities of hmos corresponding
with a small increase in fecal bacteroides sp . population ( figure 1a , b ) .
members of bacteroides and bifidobacterium sp . are known to be strong consumers of hmos .
illumina
sequencing shows a constant presence of low levels of bifidobacterium sp . in the first 3 weeks that is
not detected in the pyrosequencing results .
interestingly , by week
13 , the bacteroides are replaced by bifidobacterium sp . , which dominate the fecal bacterial
population , and
infant a microbial population changes over the first 13
weeks based
on next - generation sequencing .
( a ) illumina and ( b ) pyrosequencing
profile of the v4 and the v1v3 regions of the 16s rrna gene ,
respectively . the letters preceding the taxon are taxonomy identifiers :
p ( phylum ) , c ( class ) , o ( order ) , f ( family ) , and g ( genus ) .
bifidobacterium - specific trflp and
qpcr revealed a succession of bifidobacterial species over time ( figure 3 ) .
infantis ) dominated these samples from weeks 013 ,
followed by an increase in b. adolescentis , b. bifidum , and b.
breve at weeks 1326 ( data not shown for weeks
> 13 ) .
infant a microbial population changes over the first 13 weeks based
on qpcr and bif - trflp analyses .
qpcr of ( a ) total bacteria , bifidobacteria , and bacteroidales and ( b ) species - specific bifidobacteria populations ; ( c ) bif - trflp profile of the total bifidobacteria community .
the correlation of bifidobacterial
growth with hmo consumption
was confirmed by calculating pearson product moment correlation
coefficients between normalized hmo d / h ratios and the relative abundances
of all order - level bacterial taxa detected in at least three samples .
bifidobacteriales was the only clade yielding a significant ,
negative correlation coefficient with five hmos , as shown in table 2 ( negative correlation implying that as fecal bifidobacteria
numbers increase , fecal hmos decrease , consistent with consumption
of the hmos ) .
other bacteria demonstrate a significant , positive correlation ,
indicating that as these microbes increased fecal hmos increased as
well ( consistent with lack of consumption of hmos by these bacteria ,
e.g. , enterobacteriales and bacillales ) . a second infant , infant
b , also shows development of a saccharolytic microbiota .
figure 4a , b shows the glycoprofiles ( analyzed by maldi ft - icr
ms ) and bacterial populations ( analyzed by 16s rdna pyrosequencing )
over the infant s first 14 weeks of life , respectively .
we
observe the fecal glycans to increase dramatically from week 0 to
week 1 .
note that the y axis in this figure is logarithmic
and that the increases vary from 14-fold to > 1 million - fold . at
week
2
, there is little change in hmo abundance in the feces , whereas at
week 14 , all of the measured fecal hmos have decreased ( range 2-fold
to 7000-fold higher than baseline ) .
changes in oligosaccharide intensities
and bacterial population
in the feces of infant b. ( a ) percent change in h / d ratios of hmos
in the feces of infant b at weeks 0 , 1 , 2 , and 14 .
intensities were
obtained using maldi ft - icr ms in ( + ) ion mode with n
( b ) corresponding fecal
bacterial population of infant b using 16s rdna pyrosequencing .
s
samples yielded 30 886 and 447 149 partial 16s rrna
gene sequence reads , respectively .
order level fecal populations are
shown in figure 5a , and genus level , in figure 5b .
the fecal bacterial population of this infant
is initially populated by nearly equal amounts of enterobacteriaceae ( primarily escherichia ) and bacteroides in week 0 . by week 1
, escherichia increases to be the dominant species
as the amount of bacteroides decreases .
at week 2
, there is little change in either the fecal microbiota or
the fecal hmo composition . by week 14
, bacteroidales dominate the population at 60% , and the corresponding hmo fecal
profile shows a decrease in intensity of nearly all hmo compositions .
these results correlate with our previous in vitro observations that
members of bacteroidaceae , e.g. , bacteroides fragilis , are consumers of hmos .
infant b microbial population changes over the first 14
weeks of
life based on next - generation sequencing .
( a ) illumina and ( b ) pyrosequencing
profile of the v4 region and v1v3 regions of the16s rrna gene ,
respectively .
interestingly , the drop
in abundances of oligosaccharide for infant
b is not as large as that for infant a , where the majority of bacteria
at week 13 was bifidobacterium sp .
in vitro studies using strains of both bifidobacterium
longum ssp .
infantis ( b. infantis ) and bacteriodes thetaiotaomicron show that the former is a stronger consumer of hmo than the latter.bifidobacterium sp .
populations were
low in infant b , detectable by illumina sequencing ( figure 5a ) but not by pyrosequencing ( figure 5b ) or qpcr ( figure 6 ) .
b. longum group was the only clade detected by qpcr ,
although higher populations of b. bifidum were detected by trflp as well as small populations of b. breve and b. adolescentis ( data not shown ) .
infantb microbial population changes over the first 14
weeks of
life based on qpcr analysis .
* bifidobacteria are below the limit of detection ( 1 10 16s rrna
genes per gram of stool ) .
a more
extensive analysis supports the hypothesis of structure / isomer - specific
consumption of hmos in the infants gut .
the fecal profile
of infant a is dominated by bifidobacterium spp . by week 13 ( figure 1 ) , and the fecal
profile of infant b is dominated by bacteroides spp . by week 14 ( figure 4 ) .
although the
patterns are not equivalent , suggesting differential consumption ,
there are some similarities in the consumption or nonconsumption of
certain hmos . in the fecal profile of infant a , for example ,
a pentasaccharide with monoisotopic mass of 855.3220 having the composition
3 hexose ( galactose or glucose ) , 1 n - acetylglucosamine ,
and 1 fucose was found to have four isomers corresponding to the common
name lacto - n - fucopentaose : lnfp i , lnfp ii , lnfp
iii , and lnfp v. the fecal profiles are shown in figure 7a as quantified by the h / d ratio using nano - hlpc chip / tof
ms data .
one of these four isomers behaved unlike the other three
structurally very similar isomers .
lnfp ii ( structure inset figure 7a , note the terminal -1,4-fucosylation ) continually
increased from week 0 to week 13 ( suggesting a lack of consumption
by intestinal bacteria ) .
the other three isomers , lnfp i , iii , and
v , all increased in fecal intensity during week 1 but decreased dramatically
during week 2 and stayed very low through week 13 ( suggesting consumption ) .
h / d ratios of two isomeric groups of oligosaccharides in the fecal
hmo profile of infant a. h / d ratios were calculated using nano - hlpc
chip / tof ms data .
( a ) four isomers of m / z 856 ( [ m + h ] , m = 855.3220 , second bar from the left
in each week in figure 1a ) .
( b ) three isomers
of m / z 538 ( [ m + 2h ] , z = 2 , m = 1074.3963 , fourth bar from the left
in figure 1a ) .
figure 7b demonstrates another group of
isomers from infant a with an overall hexasaccharide structure with
a common monoisotopic mass of 1074.3963 ( m / z 538 , z = 2 ) and composed of 4 hexose
and 2 n - acetylglucosamine with at least three linear
or branched structures containing terminal (1,3)- and (1,4)-galactose
residues .
all three isomers follow a similar profile increasing in
week 1 and decreasing thereafter ( consistent with consumption ) .
lc ms
extracted ion chromatograms of m / z 538 ( [ m + 2h ] , z = 2 , m
= 1074.3963 ) in the fecal hmo profile of infant a using nano - hlpc
chip / tof ms .
chromatograms at ( a ) week 0 , ( b ) week 1 , ( c ) week 2 ,
and ( d ) week 13 .
figure 9 demonstrates marked differences
in individual fecal hmo structures of infant a over time , consistent
with the hypothesized highly selective nature of consumption of hmos
by the intestinal bacteria .
i , mflnh
iii , iflnh i , and iflnh iii ( mw 1220.4542 ) ) are present in the feces
until week 2 but diminish rapidly by week 13 ( concurrent with the
bloom of bifidobacteria ) , suggesting preferential consumption ; meanwhile ,
isomer 4120a increases at week 13 , suggesting that the fecal microbes
may be unable to digest this structure ( figure 9a ) .
isomers dflnhb , and dflnhc ( mw 1366.512 ) both show a decrease
concomitant with the bloom of bifidobacteria at week 13 ; however ,
this pattern is not seen with isomer dflnha ( figure 9b ) .
isomers 5130a , 5130b , f - lno , and 5130c ( mw 1585.5864 )
all diminished at week 13 , suggesting consumption ( figure 9c ) .
isomers 5230a , 5230b , and dflnno ii ( mw 1731.6443 )
demonstrate the pattern consistent with consumption , but dflnno i
is markedly increased , suggesting a lack of consumption by the fecal
microbes at 13 weeks ( figure 9d ) .
h / d ratios
of four isomeric groups of oligosaccharides in the fecal
hmo profile of infant a. h / d ratios were calculated using nano - hlpc
chip / tof ms data . ( a ) five isomers of m / z 611 ( [ m + 2h ] , z = 2 , m = 1220.4542 ,
sixth bar from the left in each week in figure 1a ) .
( b ) three isomers of m / z 684
( [ m + 2h ] , z = 2 , m = 1366.5121 , seventh
bar from the left in figure 1a ) .
( c ) four isomers
of m / z 794 ( [ m + 2h ] , z = 2 , m = 1585.5864 , tenth bar from the left
in figure 1a ) .
( d ) five isomers of m / z 867 ( [ m + 2h ] , z = 2 , m = 1731.6443 , 11th bar from the left in figure 1a ) .
isomer analyses of fecal hmos for infant b as quantified
by the
h / d ratios using nano - hplc chip / tof ms data are shown in supporting information figures 13 .
fecal
profiles of infant b for lacto - n - fucopentaose isomers
( mw 855.3220 ) are shown in supporting information
figure 1a .
the -1,4-fucosylated lnfp ii increased slightly
from week 0 to week 1 and then plateaued in weeks 214 ( 5.7753
0.3185 ) , suggesting nonconsumption by intestinal bacteria .
the other three isomers , lnfp i , iii , and v , increased dramatically
in fecal intensity during week 1 but decreased in intensity at week
14 , suggesting consumption at the fecal profile shift to bacteroides spp . at week 14 .
supporting information figure 1b shows
fecal profiles of another group of isomers with mw 1074.3963 .
all
three isomers , lnh , lnnh , and p - lnh , show a decrease concomitant with
the bloom of bacteroides at week 14 , suggesting consumption .
the extracted
ion chromatograms of said isomers are shown in supporting information figure 2 .
all isomers increased from
week 0 to week 2 and then decreased in week 14 , consistent with consumption .
similar to infant a , the results suggest little variation between
-galactosidase linkage isomers in infant b. other individual
fecal hmo structures and isomers are shown in supporting information figure 3 .
i , mflnh iii , iflnh
i , and iflnh iii ) decrease over time ( supporting
information figure 3a ) .
isomers dflnh a , b , and c all show
a decrease at week 14 ( supporting information
figure 3b ) .
isomers 5130a and 5130c ( mw 1585.5864 ) both show
a decrease at week 14 , suggesting consumption , whereas 5130b and f - lno
increased at week 14 ( supporting information figure
3c ) .
isomers 5230a and dflnno i and ii ( mw 1731.6443 ) show
a pattern consistent with consumption , but 5230b is markedly increased ,
suggesting a lack of consumption by the fecal microbes at 14 weeks
( supporting information figure 3d ) .
quantitation of milk oligosaccharides
is complicated
by the different ionization properties of the different structures .
suppression effects are observed when the sample is analyzed as a
mixture by mass spectrometry .
for example , sialylated oligosaccharides
are often suppressed by neutral oligosaccharides in the positive mode ,
whereas the reverse happens in the negative mode .
separation of the oligosaccharides eliminates suppression
effects ; however , there are still slight variations in the ionization
of the individual structures .
in addition , the internal standards from
the pooled milk sample contain more structures than any individual
specimen , allowing the analysis of large biological diversity .
the two infants studied were both healthy , breast - fed infants ,
but the colonization of their intestinal tract differed remarkably .
the fecal microbiota of infant a was initially dominated by streptococcus spp . , whereas the gut microbiota of
infant b was dominated by escherichia spp .
these bacteria are common commensals of the birth canal and
are not hmo consumers , i.e. , do not have the genetic capability to
produce the glycosidases necessary to break down the linkages of hmo .
the increase in fecal hmos in weeks 02 in both infants is
consistent with the hypothesized lack of hmo consumption during this
period .
however , by week 13 , bifidobacterium spp . dominated in infant a , and levels of most fecal hmos dropped
dramatically . the decline in hmo intensity is significantly negatively
correlated to bifidobacteriales abundance
and positively correlated with several other bacteria , notably , enterobacteriales and bacillales . for infant b , bacteroides spp . were
dominant by week 14
, at which time levels of many fecal hmos were
decreased compared to those at weeks 1 and 2 . the enrichment
of bifidobacterium spp . and bacteroides spp
moreover , metagenomic analysis has revealed that this enrichment
also corresponds with increased expression of many genes involved
in consumption of complex oligosaccharides .
we have previously shown in vitro that bacteroides are good consumers of hmos and that b. infantis can grow with hmos as the sole carbon
source . in spite of the complexity of
this ecological niche ,
the in vivo data here presented match closely
the in vitro results for the single species analysis .
these results
further support the concept that one function of hmos is to selectively
enrich a saccharolytic bacterial consortium despite the variety of
bacteria introduced into the infant in the early days of life .
our method allows
simultaneous analysis of up to 75 structurally
elucidated oligosaccharides and the monitoring of hundreds of structures
simultaneously .
profiles of human milk
across 6 months of lactation show the constant delivery of hmos with
minor fluctuation particularly for the smaller , more abundant oligosaccharides
( degree of polymerization < 7 ) .
this
level of detail allows the elucidation of gross glycosidic enzyme
actions by the microbiota , i.e. , the consumption of known structures
is an indicator of the presence of specific active bacterial enzymes
in the gut .
the whole genome sequencing of b.
infantis revealed a number of exoglycosidases including
galactosidases , fucosidases , and sialidases .
this high degree of enzymatic activity is not seen in several other
bifidobacterial species and in part explains the dominance of the b. longum group in infant a ( b. infantis is a member of the b. longum group ) . of particular interest is the catabolism of oligosaccharides containing
( 1,2)-fucose .
the presence of 2fl and other ( 1,2)-fucose - containing
hmos such as lacto - n - fucopentaose ( lnfp ) indicates
that the mother has at least one functional fucosyl transferase ( fut2 )
allele and is therefore a secretor ( i.e. , able to express specific
fucosylated structures in secretions such as tears , milk , or saliva ) . in adults ,
secretor status is
associated with increased fecal bifidobacteria and with alterations in susceptibility to viral and bacterial
infections .
the observation that these special
fucosylated oligosaccharides are preferentially consumed in healthy
infants suggests the hypothesis that certain bifidobacteria thrive
in a milieu that contains either milk from a secretor mom or intestinal
secretions from a secretor infant .
this may have particular value
to the fragile premature infant where human milk is partially protective
against sepsis and necrotizing enterocolitis and nonsecretor status may predispose to these common complications .
the analysis of fecal samples from two
infants was designed as
a proof - of - concept exercise to demonstrate the potential utility of
analyzing the fecal microbiota and fecal glycomics in tandem .
the
small sample size was helpful in generating hypotheses , but future
studies with larger sample sizes would be necessary to confirm and
replicate the findings .
analysis of milk specimens from the
mothers of these infants would have been helpful to confirm consistent
delivery of hmos over time ( demonstrated in other mothers , but not
in these two cases ) .
inclusion of maternal dietary analysis and details
regarding the mothers health status ( e.g. , obesity , diabetes ,
medications ) would have been useful .
the major strength of these observations
is the detailed analyses performed including differentiation of isomers
with differing patterns of possible consumption by the intestinal
microbes .
diagnostic testing of the stool of infants , children , and
adults for rapid determination of secretor status or to identify patterns
of hmos or bacterial enzymes suggestive of increased risk for necrotizing
enterocolitis , clostridium difficile colitis , or inflammatory diseases of the gut may be possible with
approaches similar to those demonstrated herein
this
proof - of - concept study combines glycomics and genomics to
monitor the changes in hmos and gut microbiota in breastfeeding infants .
the observed changes over time in two infants suggest the need for
more extensive studies of a larger cohort to test the hypothesis that
hmos enrich a cognate oligosaccharide - consuming microbial population
in the infant gut by orchestrating a shift in the infant fecal microbiota
from a nonsaccharolytic population dominated by commensals of the
birth canal to a population dominated by saccharolytic microbes concurrent
with a decrease in fecal hmo intensities
. further studies in nonhuman
primates and other mammals would be valuable to determine whether
feedback evolutionary mechanisms link the infant gut and maternal
milk oligosaccharide production . | human
milk oligosaccharides ( hmos ) play a key role in shaping and
maintaining a healthy infant gut microbiota .
this article demonstrates
the potential of combining recent advances in glycomics and genomics
to correlate abundances of fecal microbes and fecal hmos .
serial fecal
specimens from two healthy breast - fed infants were analyzed by bacterial
dna sequencing to characterize the microbiota and by mass spectrometry
to determine abundances of specific hmos that passed through the intestinal
tract without being consumed by the luminal bacteria . in both infants ,
the fecal bacterial population shifted from non - hmo - consuming microbes
to hmo - consuming bacteria during the first few weeks of life .
an initial
rise in fecal hmos corresponded with bacterial populations composed
primarily of non - hmo - consuming enterobacteriaceae and staphylococcaeae .
this was followed
by decreases in fecal hmos as the proportion of hmo - consuming bacteroidaceae and bifidobacteriaceae increased .
analysis of hmo structures with isomer differentiation
revealed that hmo consumption is highly structure - specific , with unique
isomers being consumed and others passing through the gut unaltered .
these results represent a proof - of - concept and are consistent with
the highly selective , prebiotic effect of hmos in shaping the gut
microbiota in the first weeks of life .
the analysis of selective fecal
bacterial substrates as a measure of alterations in the gut microbiota
may be a potential marker of dysbiosis . |
autism and autism spectrum disorder ( asd ) are commonly used terms for a group of
neurodevelopmental disorder that represents social deficits , communication difficulties ,
stereotyped or repetitive behaviors and cognitive delays1 , 2 . in general
, the child with
autism has not exhibited same symptom , but features very different type of autism
characteristics .
since the child with autism does not appears same or a single symptom ,
newer term of asd has been represented as a single diagnostic category of autism
characteristic linking various conditions .
it is known that asd reveals substantial genetic
variants , particularly fragile x syndrome ( fxs ) , rett syndrome , tuberous sclerosis complex
and structural chromosomal variations confer a high risk for asd3 .
fxs is the most frequent form of inherited intellectual
mental retardation and commonly known as monogenic type of autism , which shows clinical
behavioral features such as mental retardation , learning disorder , attention deficit
disorder , hyperactivity disorder , anxiety and epilepsy4,5,6 .
particularly , the individuals with fxs often exhibit characteristic
of asd or autistic behavior7 .
fxs is a
genetic disorder that occurs by mutation of fragile x mental retardation 1
( fmr1 ) gene on the x chromosome .
mutation at this site is related with
loss of fragile x mental retardation protein ( fmrp ) that affecting physical and behavioral
abnormalities .
the individuals with fxs also present with disturbed sleep and altered
circadian behavior as well . in molecular studies ,
the absence of fmr1 and
fmr2 gene induces the altered expression of clock gene related components
and changes circadian rhythm .
in addition , clinical studies reported that sleep and
behavioral alteration of fxs would be associated with fmr1 and
fmr2 gene .
as described above , asd and fxs exhibit various clinical features including mental
retardation and epileptic episodes4 , 6 .
recently , numerous studies have focused on
understanding the pathophysiological mechanisms responsible for asd and fxs , and on
developing more effective therapeutic treatments .
sairanen m et al.8 reported that asd is
characterized by structural defects that include a reduction in forebrain volume and
disruption of the neural network between the limbic system and other cerebral cortex
regions .
in addition , recent study demonstrated that asd children presented an abnormal gait
compared with that of age - matched controls , they showed a reduction in cadence , gait
velocity , step length , and an increase in step width9 .
interestingly , therapeutic physical exercise in patients with asd
increases hippocampal volume and promotes the robust growth of newly proliferated and/or
differentiated cells by increasing brain - derived neurotrophic factor ( bdnf ) in the cerebral
cortex10 .
also , the
fmr1 gene was shown to play a critical role in the neural plasticity of
dendritic spines in studies on neuropsychological function in asd and fxs .
therefore , the
absence of fmr1 results in dysmorphology and dendritic spine dysfunction11 , 12 .
in addition , fmr1 knockout mice swim significantly slower than
controls and take more time to arrive at the target platform in the morris water maze13 .
therapeutic physical exercise plays a
critical role in neuronal cell survival in asd patients , and forced aerobic exercise during
light photoperiods promotes vascularization and neurogenesis14,15,16 .
therapeutic physical exercise facilitates the production of
neurotrophic factors , including nerve growth factor ( ngf ) , fibroblast growth factor-2
( fgf-2 ) as well as bdnf , that improve neuropsychological function in infants with asd17 , 18 . in a previous study using the valporic acid ( 400 mg / kg)-induced asd
animal model , the animals with intervention of physical exercise showed incremental
improvement in spatial learning memory , decision making , and neurogenesis in the hippocampal
region via stimulating the pi3 k / akt / erk signaling pathway19
. moreover , exercise that combines physical and mental activities
has improved cognitive ability in individuals with asd , and participation of people with asd
in these activities serves the additional purpose of decreasing stereotypes20 , 21 .
strikingly , a recent report suggested that a mind - body exercise
program could positively affect neural functional connectivity and memory processing , and
could therefore enhance memory function in individuals with asd22 .
in a study that examined the relationship between stress and exercise , one patient with
autism showed abnormally high epinephrine , norepinephrine , and cortisol levels during
maximal and submaximal treadmill exercise compared to resting levels . even though
therapeutic physical exercise is somehow stressful in patient , but the subjected patient was
able to complete the entire protocol without experiencing any maladaptive behavior .
therefore , additional studies are needed to determine the beneficial effects of therapeutic
physical exercise , and to provide the clinical application guideline of exercise for
autistic fxs patients .
| [ purpose ] the purpose of the present review is to discuss recent published articles in
the understanding of efficacy of interventional exercise on autistic fragile x syndrome
( fxs ) with special emphasis on its significance in clinical application in patients .
[ methods ] this review article was identified scientifically and/or clinically relevant
articles from pubmed that directly / indirectly met the inclusion criteria .
[ results ]
mutation of fragile x mental retardation 1 ( fmr1 ) gene on the x
chromosome is related with loss of fragile x mental retardation protein ( fmrp ) that
affecting physiological and behavioral abnormalities .
autistic fxs individuals exhibit
disturbed sleep and altered circadian behavior .
although the underlying molecular
mechanisms are not been fully explored , interventional exercise in autistic fxs has been
clinically used for the treatment of physiological and behavioral abnormalities as well as
psychiatric disorder in autistic fxs .
[ conclusion ] this review describes beneficial
efficacy of interventional exercise and its controversy in patients with autistic fxs .
this review also provides interventional strategies for clinicians and scientists that the
way of neurophysiological approaches according to the level of physical and behavioral
abnormalities . |
from july 2005 through october 2006 , a survey was conducted on 194 isolates of m. tuberculosis obtained from 194 patients with pulmonary tuberculosis ( tb ) ( 1 isolate per patient ) ( 1 ) .
patients were recruited from the national hospital for pneumology and phtisiology in cotonou , benin , where most tb patients from the area are treated .
cotonou is the largest city in benin , with a population of 655,000 in 2002 and an area of 79 km .
the study was approved by the national tuberculosis program board of benin . among these 194 isolates
, 17 belonged to the beijing st1 family and exhibited the same 12-loci miru - vntr pattern ( 223325163533 ) .
the median age of the patients infected with the m. tuberculosis strains belonging to the beijing genotype ( 28 years ) was similar to that of patients from the general survey ( 30 years ) .
demographic data that included date of birth , age , sex , and places of residence and work were collected from each patient .
geo - coordinates of each patient s residence and workplace were obtained by using the global positioning system ( gps ) and mapped with the arcview 3.2 software ( esri , redlands , ca , usa ) .
seropositive samples were confirmed by a discriminatory hiv1/2 test ( genie ii hiv1/hiv2 ; bio - rad , marnes - la - coquette , france ) .
one sputum sample from each patient was decontaminated by using the modified petroff method and cultured in manual mycobacteria growth indicator tube ( mgit ) ( 6 ) and on lwenstein - jensen ( lj ) medium .
all isolates were identified as m. tuberculosis complex by the para - nitrobenzoic acid method and tested for drug susceptibility against rifampin , isoniazid , streptomycin , and ethambutol by using the proportion method on lj medium at the following respective concentrations : 40 g / ml , 0.2 g / ml , 4 g / ml , and 2 g / ml ( 7,8 ) .
dna was extracted by boiling a suspension of 2 drops of mgit - positive cultures in 300 l of 10 mmol / l tris - hcl and 1 mmol / l edta , ph 8.0 ( 1 tris - edta ) for 5 minutes .
miru - vntr typing was performed at genoscreen ( lille , france ) by amplifying each of the 24 independent loci , and results were combined into digit allelic profiles ( 5 ) .
all patients discussed here were born in benin and had lived in the country since birth .
however , because some inhabitants of cotonou are immigrants from the asian continent , this strain could have been brought into the country by migrant residents of the community . in total , 6 ( 35% ) of 17 patients were hiv-1 seropositive , and the remaining 11 patients ( 65% ) were hiv seronegative .
in contrast with the results in the initial survey , the m. tuberculosis beijing strain was more likely to be isolated from hiv seropositive patients than from those who were hiv seronegative ( 21 [ 12% ] of 173 ( p = 0.024 ) .
of the 17 m. tuberculosis isolates belonging to the beijing genotype , drug susceptibility testing results were available for 16 .
all isolates from the 16 patients were resistant to streptomycin but susceptible to isoniazid , rifampin , and ethambutol . of the remaining 177 isolates from the same survey ,
drug susceptibility testing was available for 127 , of which only 13 ( 10.2% ) were resistant to streptomycin .
the m. tuberculosis beijing strains in this survey were more likely to be resistant to streptomycin than were their non - beijing counterparts ( p<0.001 ) .
other researchers have suggested that m. tuberculosis beijing strains may be associated with drug resistance ( 9 ) and that the rapid spread of beijing strains in some settings suggests an intrinsic virulence of this family ( 10 ) .
the combination of double alleles in several miru - vntr loci of 1 isolate suggests a mixed infection ( 11 ) .
the other 16 strains showed identical profiles in the 24 miru - vntr set : loci 154 , 424 , 577 , 580 , 802 , 960 , 1644 , 1955 , 2059 , 2163b , 2165 , 2347 , 2401 , 2461 , 2531 , 2687 , 2996 , 3007 , 3171 , 3192 , 3690 , 4052 , 4156 , and 4348 with the following profile : 244233342xx4425163353723 .
no amplification was achieved for loci 2163b and 2165 , despite 2 rounds of pcr for all 16 isolates . this common failure to amplify loci 2163b and 2165 , which are close in the m. tuberculosis chromosome ,
might be explained by a chromosomal deletion in the strain responsible for this possible outbreak . having a chromosomal deletion supports the hypothesis that all patients were infected by the same strain .
however , other possible explanations such as nucleotide polymorphisms in the sequence complementary to pcr primers could not be excluded .
many studies have reported pseudo - outbreaks caused by laboratory cross - contamination ( 12,13 ) .
this is not likely in the present study because almost all the specimens for primary culture were processed on different days over several months .
mapping of patients residences and workplaces showed that many lived or worked ( or both ) in the xwlacodji area of cotonou ( figure ) . to further investigate this spatial cluster and a possible link between patients
, we mapped a place they habitually frequented and found that most patronized the same drinking bar ( figure ) .
ten of the patients either lived or worked near the bar ( < 300 m ) or regularly visited it . although no epidemiologic link was evident between these patients and the remaining 7 patients , most of the latter were motorcycle taxi drivers and regularly moved from place to place .
maps showing residences and workplaces of mycobacterium tuberculosis patients in xwlacodji , cotonou , benin , 20052006 . without gps
we could not have identified the geographic cluster of these patients with beijing strains in xwlacodji .
the residents are poor , and overcrowding creates conditions favorable for transmission of diseases from person to person and rapid spread of the resistant m. tuberculosis beijing strain .
we can not completely exclude the unlikely possibility that the patients were infected by different strains with the same miru - vntr pattern .
however , the 17 strains identical 24-loci miru - vntr profile , with a probable deletion of the same 2 loci ; the identical drug susceptibility pattern ( monoresistant to streptomycin ) ; and the fact that most patients resided in the same community as determined by gis strongly suggest that these strains are part of an outbreak .
generally , molecular tools are used to study tb transmission and to suggest possible outbreaks .
gis has rarely been used in health systems , particularly in resource - poor countries such as benin ( 14 ) .
gis tools might be dispensable in industrialized countries where streets and houses are properly mapped and pinpointing specific addresses is generally sufficient to localize an outbreak . in many low - income settings , however , streets and houses are not properly numbered and mapped , and using gps is necessary to gain access to accurate geo - coordinates and localize such outbreaks . in the absence of genetic methods , differentiating cases from the same route of transmission from others
however , gis can help tb control practitioners identify areas with aggregate cases so they can institute appropriate measures to control the disease . | using geographic information system and molecular tools , we characterized a possible outbreak of tuberculosis caused by mycobacterium tuberculosis beijing strain in 17 patients in cotonou , benin , during july 2005october 2006 .
most patients lived or worked in the same area and frequented the same local drinking bar .
the isolates were streptomycin resistant . |
caebv infection was defined as 1 ) persistent or recurrent symptoms related to ebv infection for more than three months ; 2 ) high ebv genome levels in affected tissues or peripheral blood ; 3 ) chronic illness that could not be explained by other known disease processes at diagnosis , and 4 ) no specific underlying immunological abnormality.7 to identify ebv - positive systemic t - cell lpd of childhood based on the 2008 who classification , all patients with ebv - positive hemophagocytic syndrome presenting at our institution were enrolled in the present study .
patient inclusion was based on the fulfillment of the diagnostic criteria for hlh-2004 in addition to the presence of a high viral load in the tissues or peripheral blood.3 the surgical pathology database of samsung medical center from 1994 to 2012 was screened with keywords including " caebv , " " ebv , " and " hemophagocytosis . " of the 96 cases retrieved using " ebv " and " hemophagocytosis " as keywords , 84 patients were excluded because lpd had arisen in immunocompromised patients , such as those receiving organ transplantation or patients with congenital immune deficiency syndrome .
fifteen patients were diagnosed with caebv infection , and 12 patients had paraffin blocks available for examination . among the remaining 12 patients , seven had paraffin blocks available for examination and were enrolled in the present study .
formalin - fixed paraffin - embedded blocks of liver , bone marrow , and lymph node tissues were used for further investigation .
immunohistochemical analysis was performed on the paraffin sections using monoclonal and polyclonal antibodies to detect lineage - specific or lineage - characteristic antigens , including cd3 ( dakopatts , copenhagen , denmark ) , cd20 ( novocastra , newcastle upon tyne , uk ) , cd56 ( novocastra ) , cd4 ( novocastra ) , and cd8 ( novocastra ) . for the polymerase chain reaction ( pcr )
amplification of the tcr locus , dna was extracted from the paraffin - embedded formalin - fixed tissues with the qiaamp dna ffpe tissue kit ( qiagen , hilden , germany ) . conventional pcr followed by single - stranded conformational polymorphism analysis
was performed , as previously described.8 when conventional pcr for tcr genes produced negative results , further analysis was performed using the tcr and primers , according to the recommendations of the biomed-2 protocols ( invivoscribe , san diego , ca , usa).9
caebv infection was defined as 1 ) persistent or recurrent symptoms related to ebv infection for more than three months ; 2 ) high ebv genome levels in affected tissues or peripheral blood ; 3 ) chronic illness that could not be explained by other known disease processes at diagnosis , and 4 ) no specific underlying immunological abnormality.7 to identify ebv - positive systemic t - cell lpd of childhood based on the 2008 who classification , all patients with ebv - positive hemophagocytic syndrome presenting at our institution were enrolled in the present study .
patient inclusion was based on the fulfillment of the diagnostic criteria for hlh-2004 in addition to the presence of a high viral load in the tissues or peripheral blood.3 the surgical pathology database of samsung medical center from 1994 to 2012 was screened with keywords including " caebv , " " ebv , " and " hemophagocytosis . " of the 96 cases retrieved using " ebv " and " hemophagocytosis " as keywords , 84 patients were excluded because lpd had arisen in immunocompromised patients , such as those receiving organ transplantation or patients with congenital immune deficiency syndrome .
fifteen patients were diagnosed with caebv infection , and 12 patients had paraffin blocks available for examination . among the remaining 12 patients , seven had paraffin blocks available for examination and were enrolled in the present study .
formalin - fixed paraffin - embedded blocks of liver , bone marrow , and lymph node tissues were used for further investigation .
immunohistochemical analysis was performed on the paraffin sections using monoclonal and polyclonal antibodies to detect lineage - specific or lineage - characteristic antigens , including cd3 ( dakopatts , copenhagen , denmark ) , cd20 ( novocastra , newcastle upon tyne , uk ) , cd56 ( novocastra ) , cd4 ( novocastra ) , and cd8 ( novocastra ) .
for the polymerase chain reaction ( pcr ) amplification of the tcr locus , dna was extracted from the paraffin - embedded formalin - fixed tissues with the qiaamp dna ffpe tissue kit ( qiagen , hilden , germany ) . conventional pcr followed by single - stranded conformational polymorphism analysis
was performed , as previously described.8 when conventional pcr for tcr genes produced negative results , further analysis was performed using the tcr and primers , according to the recommendations of the biomed-2 protocols ( invivoscribe , san diego , ca , usa).9
all patients ( 12 males and eight females ) were korean , ranging from 4 to 61 years of age .
tcr gene rearrangement analysis revealed monoclonality in two patients ( 28.6% ) and polyclonality in four patients ( 57.1% ) ( fig .
two patients with t - cell monoclonality were classified as suffering systemic t - cell lpd and the remaining patients as suffering ebv - positive hlh .
of the two patients with systemic t - cell lpd , one was a young female and the other an adult male .
the ebv - positive hlh patients included two children , two young adults , and one elderly adult , with ages ranging from 4 to 61 years ( median , 20 years ) .
the clinicopathological findings for the two conditions were similar , and almost all patients had acute - onset fever .
the duration of the symptoms was two months in patients with systemic t - cell lpd and ranged from three days to one month ( median , 1 month ) in ebv - positive hlh patients .
two patients with systemic t - cell lpd received chemotherapy but died after two and three months , respectively . among the five patients with ebv - positive hlh , four received chemotherapy or steroid and died after a median period of one month , whereas the one patient treated with the hlh-2004 regimen recovered completely and was alive at the final follow - up .
the ebv load in the peripheral blood was analyzed using whole blood , and the loads during the clinical course are shown in table 1 .
the levels were variable , and two patients with ebv - positive hlh showed high viral loads in their peripheral blood .
biopsies were obtained from the bone marrow of six patients , lymph nodes of two patients , liver of one patient , and spleen of one patient . in the bone marrow , liver , and spleen samples ,
hemophagocytic histiocytosis was observed in all patients . the lymph node biopsies from patient 1 ( systemic t - cell lpd ) and patient 3 ( ebv - positive hlh ) demonstrated diffuse effacement of the nodal architecture by the infiltration of monotonous medium - sized atypical lymphocytes ( fig .
at the first liver biopsy of patient 3 , ebv - infected cells were small to medium size and showed mild to equivocal atypia ( fig .
after three months , the lymph nodes of patient 1 were effaced and infiltrated by medium - sized atypical ebv - positive lymphocytes ( fig .
3c - f ) . on initial examination , the histological findings suggested neoplastic proliferation , but patient 3 displayed polyclonality according to the tcr gene rearrangement ( table 1 ) .
the bone marrow of the two patients showed similar findings to the observations in the lymph nodes .
the phenotype of the infiltrated cells was cd3-positive , cd20-negative , and cd56-negative in all cases .
ebv - positive hlh was cd8-positive t - cell dominant in three patients and cd4-positive t - cell dominant in two patients .
in situ hybridization used to detect ebv in biopsy tissues using the ebv - encoded early rna 1 probe showed the presence of ebv - infected cells in six of seven patients ( fig .
the numbers of ebv dna copies in the peripheral blood varied , ranging up to 5,810 copies/l .
the caebv group consisted of 12 patients ( eight males and four females ) , with ages ranging from 10 to 59 years ( median , 21 years ) ( table 2 ) .
nine patients were children or young adults , one patient was elderly , and their clinical manifestations varied .
mosquito - bite hypersensitivity was observed in four children , and three patients had nk lymphocytosis .
some patients manifested unusual clinical findings , such as bowel perforation , immunoglobulin a nephropathy , choreic movement , and brain infarction .
biopsies of the bone marrow , skin , gastrointestinal tract , or lung were performed .
ebv - positive lymphocytes were scattered , and infiltrated lymphocytes were predominantly small and devoid of atypia ( fig .
on immunohistochemistry , cd4-positive t - cells were dominant in three patients and cd8-positive cells were dominant in three patients .
monoclonality was detected in three of the six patients in whom pcr analysis of the tcr gene was successful . among the three polyclonal patients , one was cd56-positive , with a skewed killer cell immunoglobulin - like receptor ( kir ) phenotype , suggesting monoclonal nk - cell proliferation . among the three patients with polyclonal t - cell proliferation , two were alive and one was alive 30 months after diagnosis .
of the three patients with monoclonal t - cell proliferation , two patients with monomorphic histology died of their disease after ten months and seven months , respectively , and one patient with polymorphic histology had persistent disease six months after diagnosis .
comparison of patient survival according to clonality was not statistically meaningful because of the limited number of patients included in the study , but monoclonal patients tended to have poorer prognosis .
tcr gene rearrangement analysis revealed monoclonality in two patients ( 28.6% ) and polyclonality in four patients ( 57.1% ) ( fig .
two patients with t - cell monoclonality were classified as suffering systemic t - cell lpd and the remaining patients as suffering ebv - positive hlh .
of the two patients with systemic t - cell lpd , one was a young female and the other an adult male .
the ebv - positive hlh patients included two children , two young adults , and one elderly adult , with ages ranging from 4 to 61 years ( median , 20 years ) .
the clinicopathological findings for the two conditions were similar , and almost all patients had acute - onset fever .
the duration of the symptoms was two months in patients with systemic t - cell lpd and ranged from three days to one month ( median , 1 month ) in ebv - positive hlh patients .
two patients with systemic t - cell lpd received chemotherapy but died after two and three months , respectively . among the five patients with ebv - positive hlh , four received chemotherapy or steroid and died after a median period of one month , whereas the one patient treated with the hlh-2004 regimen recovered completely and was alive at the final follow - up .
the ebv load in the peripheral blood was analyzed using whole blood , and the loads during the clinical course are shown in table 1 .
the levels were variable , and two patients with ebv - positive hlh showed high viral loads in their peripheral blood .
biopsies were obtained from the bone marrow of six patients , lymph nodes of two patients , liver of one patient , and spleen of one patient . in the bone marrow , liver , and spleen samples ,
the lymph node biopsies from patient 1 ( systemic t - cell lpd ) and patient 3 ( ebv - positive hlh ) demonstrated diffuse effacement of the nodal architecture by the infiltration of monotonous medium - sized atypical lymphocytes ( fig .
at the first liver biopsy of patient 3 , ebv - infected cells were small to medium size and showed mild to equivocal atypia ( fig .
after three months , the lymph nodes of patient 1 were effaced and infiltrated by medium - sized atypical ebv - positive lymphocytes ( fig .
the histological findings suggested neoplastic proliferation , but patient 3 displayed polyclonality according to the tcr gene rearrangement ( table 1 ) .
the bone marrow of the two patients showed similar findings to the observations in the lymph nodes .
the phenotype of the infiltrated cells was cd3-positive , cd20-negative , and cd56-negative in all cases .
ebv - positive hlh was cd8-positive t - cell dominant in three patients and cd4-positive t - cell dominant in two patients .
in situ hybridization used to detect ebv in biopsy tissues using the ebv - encoded early rna 1 probe showed the presence of ebv - infected cells in six of seven patients ( fig .
2 ) . in one patient , in situ hybridization failed to detect ebv because the tissue was improperly fixed .
the numbers of ebv dna copies in the peripheral blood varied , ranging up to 5,810 copies/l .
tcr gene rearrangement analysis revealed monoclonality in two patients ( 28.6% ) and polyclonality in four patients ( 57.1% ) ( fig .
two patients with t - cell monoclonality were classified as suffering systemic t - cell lpd and the remaining patients as suffering ebv - positive hlh .
of the two patients with systemic t - cell lpd , one was a young female and the other an adult male .
the ebv - positive hlh patients included two children , two young adults , and one elderly adult , with ages ranging from 4 to 61 years ( median , 20 years ) .
the clinicopathological findings for the two conditions were similar , and almost all patients had acute - onset fever .
the duration of the symptoms was two months in patients with systemic t - cell lpd and ranged from three days to one month ( median , 1 month ) in ebv - positive hlh patients .
two patients with systemic t - cell lpd received chemotherapy but died after two and three months , respectively . among the five patients with ebv - positive hlh , four received chemotherapy or steroid and died after a median period of one month , whereas the one patient treated with the hlh-2004 regimen recovered completely and was alive at the final follow - up .
the ebv load in the peripheral blood was analyzed using whole blood , and the loads during the clinical course are shown in table 1 .
the levels were variable , and two patients with ebv - positive hlh showed high viral loads in their peripheral blood .
biopsies were obtained from the bone marrow of six patients , lymph nodes of two patients , liver of one patient , and spleen of one patient . in the bone marrow , liver , and spleen samples ,
hemophagocytic histiocytosis was observed in all patients . the lymph node biopsies from patient 1 ( systemic t - cell lpd ) and patient 3 ( ebv - positive hlh ) demonstrated diffuse effacement of the nodal architecture by the infiltration of monotonous medium - sized atypical lymphocytes ( fig .
at the first liver biopsy of patient 3 , ebv - infected cells were small to medium size and showed mild to equivocal atypia ( fig .
after three months , the lymph nodes of patient 1 were effaced and infiltrated by medium - sized atypical ebv - positive lymphocytes ( fig .
, the histological findings suggested neoplastic proliferation , but patient 3 displayed polyclonality according to the tcr gene rearrangement ( table 1 ) .
the bone marrow of the two patients showed similar findings to the observations in the lymph nodes .
the phenotype of the infiltrated cells was cd3-positive , cd20-negative , and cd56-negative in all cases .
ebv - positive hlh was cd8-positive t - cell dominant in three patients and cd4-positive t - cell dominant in two patients .
in situ hybridization used to detect ebv in biopsy tissues using the ebv - encoded early rna 1 probe showed the presence of ebv - infected cells in six of seven patients ( fig .
2 ) . in one patient , in situ hybridization failed to detect ebv because the tissue was improperly fixed .
the numbers of ebv dna copies in the peripheral blood varied , ranging up to 5,810 copies/l .
the caebv group consisted of 12 patients ( eight males and four females ) , with ages ranging from 10 to 59 years ( median , 21 years ) ( table 2 ) .
nine patients were children or young adults , one patient was elderly , and their clinical manifestations varied .
mosquito - bite hypersensitivity was observed in four children , and three patients had nk lymphocytosis .
some patients manifested unusual clinical findings , such as bowel perforation , immunoglobulin a nephropathy , choreic movement , and brain infarction .
biopsies of the bone marrow , skin , gastrointestinal tract , or lung were performed .
ebv - positive lymphocytes were scattered , and infiltrated lymphocytes were predominantly small and devoid of atypia ( fig .
cd4-positive t - cells were dominant in three patients and cd8-positive cells were dominant in three patients .
monoclonality was detected in three of the six patients in whom pcr analysis of the tcr gene was successful . among the three polyclonal patients , one was cd56-positive , with a skewed killer cell immunoglobulin - like receptor ( kir ) phenotype , suggesting monoclonal nk - cell proliferation . among the three patients with polyclonal t - cell proliferation , two were alive and one was alive 30 months after diagnosis .
of the three patients with monoclonal t - cell proliferation , two patients with monomorphic histology died of their disease after ten months and seven months , respectively , and one patient with polymorphic histology had persistent disease six months after diagnosis .
comparison of patient survival according to clonality was not statistically meaningful because of the limited number of patients included in the study , but monoclonal patients tended to have poorer prognosis .
ebv - positive hlh is a clinical entity described in the literature that includes a wide spectrum of illnesses , ranging from ebv - associated reactive polyclonal lymphoproliferation to monoclonal disease.10 - 12 ebv - associated hlh can be divided into three evolutional stages . the innate immune response phase in the first week of ebv infection is followed by the t - cell activation stage in the second week , which is followed later by the macrophage activation stage . during the last evolutionary phase
, a substantial percentage of patients may progress to monoclonal t - cell lpd with or without an abnormal karyotype , which is equivalent to ebv - positive systemic t - cell lpd of childhood.13,14
in vitro studies have suggested that the tumor necrosis factor ( tnf ) receptor secreted by ebv - infected t - cells and the tnf - receptor - associated factors / nuclear factor-b / extracellular signal - related kinase pathway activated by the eb viral protein , lpm-1 , play a role in the progression to t - cell lymphoma.15 the " ebv - positive systemic t - cell lpd of childhood " in the 2008 who classification corresponds to the neoplastic stage of ebv - positive hlh .
however , the separation of systemic t - cell lpd from ebv - positive hlh based on clonality raises two issues : difficult diagnosis due to the technical limitations of clonality assessment , and the clinical impact of clonality on ebv - positive nk- or t - cell lpd .
the clonality of ebv - infected lpd can be determined by various methods , including tcr gene rearrangement , cytogenetic studies , and investigation of the terminal repeats of the ebv genome .
tcr gene rearrangement , detected by conventional pcr analysis or southern blot hybridization , is inadequate to examine the clonality of t - cell ebv - infected lpd because of its low sensitivity .
ebv - infected cells defined as polyclonal by tcr analysis can be monoclonal based on ebv terminal repeat analysis or cytogenetic analysis .
the sensitivity of the recently developed biomed-2 assay is superior to the conventional pcr assay.16 in addition to low sensitivity , a tcr gene rearrangement study is disadvantageous because it can not be used for ebv - positive hlh of nk - cell types .
ebv usually infects b - cells and uncommonly t - cells , but nk - cells can also be infected .
a recent study by kimura et al.17 demonstrated that a minor proportion of ebv - positive hlh is actually an nk - cell disorder .
human nk - cells use a sophisticated system of inhibitory and stimulatory receptors of the kir gene family , which are expressed in a clonally distributed manner .
nk - cell kir gene phenotyping can help determine the clonality of nk - cells,18 but fresh samples are required .
thus , this technique has limited utility in the analysis of paraffin - embedded tissues .
analysis of the ebv terminal repeat may be the most sensitive method because it can be applied in t- , b- and nk - cell disorders .
however , ebv clonality is difficult to analyze with routine diagnostic biopsies because large amounts of freshly sampled dna for southern blot hybridization are required ; chromosomal studies share the same issue .
the clonality of ebv - positive hlh has been reported in a few studies from asia.12,19,20 a study using conventional pcr reported monoclonal tcr gene rearrangements in ten of 30 adult patients and immunoglobulin gene rearrangements in eight of the same 30 patients.19 the study by imashuku et al.,12 who analyzed the ebv terminal repeat with southern blotting , revealed a monoclonal pattern in 20 of 24 patients , a biclonal pattern in two patients , and a polyclonal pattern in two patients . in their study , the tcr gene rearrangement was monoclonal in 15 of 25 patients and polyclonal in ten of the same 25 patients .
clonal cytogenetic abnormalities were observed in seven of 23 patients.12 recently , matsuda et al.20 analyzed the tcr gene with the biomed-2 protocol , the most recent and advanced technique for gene rearrangement studies and found at all six children with hlh showed a clonal band .
according to the above - mentioned studies , a significant proportion of ebv - positive hlh is a clonal disease that would be classified as stlpd of childhood by the current who classification . in previous studies that analyzed the clonality of ebv and proliferating cells in ebv - positive hlh , clonality itself apparently had no clinical impact on patient outcome.19 imashuku et al.12 reported that the clonality of t- and b - cells has no prognostic significance , whereas all patients with cytogenetic abnormalities experience an aggressive clinical course .
the clinical significance of a clonal karyotypic abnormality was also reported in other studies.21 during the transformation from benign reactive lymphoid proliferation to overt neoplastic disease , a small clone of ebv - infected t- or nk - cells may emerge11 and transform into more malignant cells as genetic changes accumulate .
early clonal lpd in this neoplastic transformation may show a similar treatment response to polyclonal ebv - positive hlh , but overt malignant lymphoma with chromosomal abnormality will behave differently . in terms of patient management ,
the clinician may use chemotherapy as the initial treatment when the patient is diagnosed with systemic t - cell lpd ; however , in patients with ebv - positive hlh , the clinical selection of a therapeutic modality depends on the risk factors for ebv - positive hlh : persistently high ebv genome copy number in the serum , acute fulminant or caebv infection , abnormal karyotype , and an association with hereditary disease.22,23 currently , successful therapies such as conservative / mild treatments without etoposide , hlh-94/2004-type treatment with etoposide , and very aggressive lifesaving measures , including hematopoietic stem cell transplantation , are administered to hlh patients,2,3 illustrating a survival rate greater than 75%.24 in our series , one child treated with the hlh-2004 regimen recovered completely and is alive , whereas other patients who received chemotherapy died .
similarly , two patients with stlpd died despite chemotherapy . because stlpd does not usually respond to chemotherapy , research for new treatments is needed .
after a primary ebv infection , the majority of patients recover completely , but a substantial proportion of patients develop caebv infection , which is currently incurable .
the clinical activity of the disease may depend on the balance between the host immune function and the virus .
the usual symptoms are fever , hepatic dysfunction , lymphadenopathy , or splenomegaly ; however , as shown in the present study , patients often present with unusual clinical manifestations , such as chorea , cerebral hemorrhage , generalized myositis , glomerulonephritis , and pulmonary hypertension.25,26 the disease develops in children and adolescents but can also develop in young adults and elderly patients , although with a lower frequency.17,27 caebv infection is almost always accompanied by varying degrees of lymphoproliferation . in the asian population ,
the clonality of ebv and ebv - infected t- or nk - cells varies and may be polyclonal , oligoclonal , or monoclonal . as the disease progresses from polyclonal lymphoproliferation to monoclonal disease ,
histological atypia increases.28 ohshima et al.28 proposed the categorization of caebv infection into three groups , polymorphous and polyclonal , polymorphous and monoclonal , or monomorphic and monoclonal , based on the clonality and histological changes . in the series reported by ohshima et al.,28 eight of 48 patients with caebv infection were polyclonal for tcr gene rearrangement , and
the infiltrated cells displayed polymorphic histomorphology ; 15 patients showed polymorphic morphology and biclonal or monoclonal tcr gene rearrangement ; and 25 patients showed monomorphic histomorphology and monoclonal tcr gene rearrangement .
patients with the monomorphic and monoclonal type of caebv infection had poorer prognosis than patients with polymorphic polyclonal or polymorphic monoclonal disease .
the survival of the polymorphic polyclonal and polymorphic monoclonal groups did not differ significantly.28 according to the 2008 who classification , systemic t - cell lpd corresponds to the polymorphic monoclonal and monomorphic monoclonal groups of caebv infection . despite the limitations of a small sample size in our study
, the patients with monoclonal caebv infection tended to have a poorer prognosis . in conclusion , ebv - positive hlh and systemic t - cell lpd share similar clinicopathological findings and
may constitute a continuous spectrum of acute ebv - associated t- or nk - cell proliferative disorders .
the distinction of ebv - positive t - cell lpd from ebv - positive hlh may be difficult during routine diagnoses due to technical limitations of clonality assessment .
the clinical impact of clonality in acute ebv - associated t- or nk - cell proliferative disorders is unclear , whereas monoclonal caebv infection disease tends to have a worse prognosis . | backgroundepstein - barr virus ( ebv)-associated hemophagocytic lymphohistiocytosis ( hlh ) , ebv - positive systemic t - cell lymphoproliferative disease ( stlpd ) of childhood , and chronic active ebv ( caebv ) infection may develop after primary ebv infection .
this study reviewed the clinicopathological spectrum of ebv - associated t- and natural killer ( nk)-cell lpd , including stlpd and caebv infection , with an analysis of t - cell clonality.methodsclinicopathological features of seven patients with ebv - associated hlh or stlpd and 12 patients with caebv infection were reviewed .
immunohistochemical staining and a t - cell receptor ( tcr ) gene rearrangement study were performed.resultsstlpd and ebv - positive hlh showed significantly overlapping clinicopathological findings .
one patient with stlpd and one patient with ebv - positive hlh demonstrated moderate to severe atypia of the infiltrating lymphocytes , whereas the remaining patients lacked significant atypia .
twelve patients had caebv infection , four of whom suffered mosquito - bite hypersensitivity , five showed nk lymphocytosis , and one suffered hydroa vacciniforme . infiltrating lymphocytes were predominantly small and devoid of atypia .
hemophagocytic histiocytosis was found in seven of 11 patients .
monoclonality was detected in three ( 50% ) of the six patients with successful tcr gene analysis.conclusionsebv-positive hlh and stlpd share similar clinicopathological findings and may constitute a continuous spectrum of acute ebv - associated t- or nk - cell proliferative disorders .
the distinction of ebv - positive t - cell lpd from ebv - positive hlh may be difficult during routine diagnoses because of the technical limitations of clonality assessment . |
the impact of hai includes prolonged hospital stay , long - term disability , increased resistance of microorganisms to antimicrobials , massive additional financial burden , high costs for patients and their families , and excess deaths [ 1 - 3 ] . in october 2005 the world health organization ( who ) started the " clean care is safer care " program to promote safe hand hygiene practices globally and at all levels of health care as a first step in ensuring high standards of infection control and patient safety .
the program provided technical recommendations and strategies to improve hand hygiene and included the development of who guidelines on hand hygiene in health care along with a package of practical tools to facilitate implementation activities at a facility level . who recommends hand rub with alcohol - based antiseptic agents as the gold standard procedure to protect patients from the multitude of harmful resistant and non - resistant organisms transmitted by health care workers ' hands , and this easy and fast ( 20 - 30 seconds ) procedure has been associated with reduction in nosocomial infections . differently ,
standard hand washing with water and soap , is indicated only in case of visibly dirty hands or infection due to spore - forming pathogens [ 6 , 7 ] .
the infection control team can use it to evaluate the effectiveness of specific interventions , as well as to introduce changes that minimize the risk of hai .
several methods have been suggested to monitor hand hygiene compliance ( direct observation , product use measurement , surveys , electronic systems ) , but the ideal one has not yet been identified [ 8 , 9 ] . who considers direct observation the most effective method to monitor the health care workers adherence to the hand hygiene recommendations , meanwhile the alcoholbased antiseptic agents consumption is a proxy indicator of hand hygiene . in 2006 ,
italy officially adopted the who program as a mainstay of its strategy for the promotion of hand hygiene in health care settings .
" istituto giannina gaslini " ( igg ) children hospital , genoa - italy , a tertiary care pediatric hospital in northern italy , complied with this program since 2007 .
the aim of this study was to present a new method to estimate and report on adherence to correct hand hygiene procedures ( chhp)introduced in our hospital since 2007 .
istituto giannina gaslini ( igg ) , genoa - italy is a tertiary care children 's hospital in northern italy serving as local pediatric hospital for the genoa area , but representing a tertiary care referring hospital for the whole italy and many foreign countries . in november 2007 , igg joined the " clean care is safer care " who program .
this was firstly applied in the pediatric intensive care unit ( picu ) , and then gradually extended to all hospital wards . from january to march 2008 , health care workers started a hand care hygiene educational program , conducted by the nurses of the igg infection control team . "
how to " and " 5 moments " posters about hand washing were displayed close to the sinks . beyond the standard antiseptic hand washing , hand decontamination with isopropyl alcohol gel was also recommended .
its use was implemented placing alcoholic gel dispenser at the ward entrance and next to each patient location .
as the program began , random audits were performed to check the proper application of the procedures ; re - training sessions and meetings with the infection control team nurses were performed when needed .
adherence to hand hygiene was initially estimated by means of the amount of antiseptic product supplied , expressed in terms of litres of isopropyl alcohol gel supplied to the unit per 1000 hospitalization - days . however , since this approach could be hard to understand for non - infection control experts , in 2010 another hand hygiene reporting method was introduced , and associated to the first one . as the amount needed for an effective hand hygiene procedure is known for each product ( manufacturer 's instructions ) , we estimated the number of the performed procedures by dividing the total volume of isopropyl alcohol gel ( hereinafter referred also as hand hygiene product ) supplied in a period ( a quarter ) by the volume of the product indicated for an effective hand hygiene procedure .
this number was then divided by 90 ( days in a quarter ) and then by the mean number of beds active in each day in the unit in that period ( average bed occupancy in a quarter ) .
this calculation should estimate the number of daily chhp procedures performed at each patient 's bed .
data about alcoholic gel consumption were collected by each unit head nurse , crossed with data from the pharmacy service supply to any given unit in a given period ( a quarter ) , and communicated to the infection control team . since 2010 the program was gradually extended and in april 2014 , after 4 years of progressive extension to different wards , the method was considered adequate to involve all igg wards . from that moment the infection control team drafted quarterly reports including both the amount of hand hygiene products used by each unit and the estimated number of chhp procedures performed . moreover ,
the presence of epidemic clusters , defined as an aggregation of cases of infection , without regard to whether the number of cases was more than expected , was evaluated and reported in real time , and it was followed by specific retraining sessions .
data were collected on an electronic spread sheet and graphically reported . in order to compare the 2 methods to report on chhp we calculated the pearson correlation coefficient that is a measure of the linear dependence between the 2 methods ( litres/1000 hospitalization days and chhp estimates ) .
graphics and correlation coefficient were obtained by means of microsoft office excel 2007 ( microsoft corporation redmond wa ) .
figure 1 presents the quarterly reports from april 2014 to march 2016 ( eight trimesters ) of nine selected unites : intensive care units [ pediatric intensive care unit ( picu ) , neonatal intensive care unit ( nicu ) ] , pulmonology , infectious diseases , hemato - oncology , hematopoietic stem cell transplantation , general surgery , neurosurgery , cardiology and cardiovascular surgery .
table i reports data on adherence to the hand hygiene program estimated according to two different methods , together with the pearson correlation coefficients between the 2 methods , and show the presence of a positive correlations between the 2 systems that described in the same way changes in procedures for hand hygiene , while figure 1 is the graphic representation of these results .
for example , during the third trimester of 2015 in picu , a cluster of acinetobacter baumannii colonization/ infection was followed by an intervention of the infection control team and we observed an increase in hand hygiene procedures compared to the previous trimester , which is reported by both methods ( 47 chhp vs. 19 and 135 litres of hand hygiene products vs 54 , respectively ) ( fig .
1a ) . during the third trimester of 2015 pulmonology ward experienced a cluster of carbapenem resistant enterobacter colonization , and after an intervention of the infection control team we observed an increase of both parameters compared to the previous trimester ( 24 chhp vs 11 and 72 litres of hand hygiene products vs 36 , respectively ) .
however , during the fourth trimester of 2015 , when the infection control team intervention became less pressing ( and maybe less persuasive ) , a reduction in the number of hand hygiene procedures ( from 24 to 12 ) was observed : this trend was considered worrisome and induced the infection control team to retrain health workers .
the result obtained in the following period ( 14 procedures per patient per day in the first trimester of 2016 ) was still considered not adequate , even if improving , so that a new educational intervention was programmed ( fig .
changes in litres of hand hygiene products/1000 hospitalization days ( l/1000 hd ) and estimated hand hygiene ( ehh ) products in different quarters in different wards . quarterly supply hand - hygiene products ( litres/1000 hospitalization days , dark - gray bars ) and estimates of hand - hygiene procedures ( light - gray bars ) and in different wards over 2 years of observation . in general , in icus ( fig . 1a , fig .
1b ) the number of chhp was generally larger compared to medical wards , like pulmonology or infectious disease ( fig .
1d ) . on the other hand , in surgical wards ( general surgery , neurosurgery , cardiovascular surgery ) ( fig .
1i ) the number of estimated procedures was quite similar , constant and relatively low , even if within the standard indicated by the who ( 20 liters/1000 hospitalization days ) , and in absence of any epidemic clusters or high levels of surgical site infections rate . in these wards , the decision was made to periodically retrain the health staff , though with quite frustrating results .
unfortunately , all the methods currently used to monitor both standard hand washing and chhp show some limits .
for example , direct observation of staff members is currently considered the gold standard in hand hygiene compliance monitoring , but it is time - consuming and requires an adequate number of monitors to be performed frequently in a large number of hospital wards in a reasonably time . on the other hand , direct observation can influence and modify behaviour of health workers when realizing that they are being observed ( so called hawthorne effect ) , leading to falsely elevated compliance rates . starting from a conventional approach , based on measuring litres of isopropyl alcohol gel consumed per 1000 hospitalization - days ,
the igg introduced a new method , still based on the amount of hand hygiene product , that estimates the mean number of daily chhp performed per patient .
our estimate of chhp can be performed using data that can be easily obtained by the hospital administration ( litres of hand hygiene products , days of hospitalization , mean number of patients present in a trimester in a given ward ) .
furthermore , the method we adopted to estimate chhp is unobtrusive and allows collecting and reporting real - life information without compartmental bias , even if it does not evaluate if the procedure of hand hygiene is performed in a correct way or not . as shown by this paper ,
these two methods provide overlapping results in terms of the description of hand hygiene performance over time .
in fact there was a very strict positive correlation between these measure methods , with a coefficient > 0.80 in all cases ( in 8/9 cases > 0.94 ) , a value that can be considered very high in social science , where there may be a greater contribution from complicating factors .
the new one provides the health staff with a ( estimated ) measure of its hand hygiene performance ( estimated mean number of chhp per patient per day ) , while the other one refers to the amount of hand hygiene products consumed in a unit during a certain period ( litres of product per 1000 hospitalization - days ) .
indeed , it is easier to understand one 's own hygiene conduct by thinking in terms of number of hand hygiene procedures than in terms of gel consumption .
figure 1 is a realistic picture of the mean daily number of chhp performed per patient per day in each unit .
noteworthy these numbers can fluctuate , sometimes widely , from one trimester to another within the same ward .
therefore , the main helpfulness of this analysis is to compare each unit against itself over time .
however , some observations arise from the comparison among the hand hygiene trend of different units . for example
, the higher number of procedures found in intensive care units compared to medical wards is probably due to their different case mix . indeed
, intensive care units usually host critical patients , who require constant assistance and frequent invasive procedures and , therefore , a higher number of chhp procedures is expected . on the other hand , the low number of procedures observed in surgical wards , even in absence of epidemic clusters or high levels of surgical site infections , can be at least partially explained by the fact that surgical patients undergoing more complex interventions are usually admitted to intensive care units during the first postoperative phases , which are the most critical periods , while they are referred to surgical wards when a lower level of assistance , and therefore of hand hygiene procedures , is needed . if we consider that the risk of carbapenem resistant enterobacteriaceae transmission can be significantly reduced by performing at least 50% of the required hand washings , it is at least partially understandable that surgical wards could need a relatively low number of procedures to prevent pathogens ' transmission , provided that the volumes of products are adequate , at least according to who standards .
these considerations well applies to our pediatric reality and its type of patients and surgical procedures , but they can not be simply transposed to adult or pediatric wards / hospitals with a different case mix . for this reason , we believe that our method of estimating chhp can be used in each setting to compare its own performance in process of time , but can be roughly used to compare different realities in different hospitals or even in the same one .
first , it estimates the adherence to chhp but does not provide the real number of performed procedures .
second , since it does not include direct observation , it does not allow fixing errors in real time , but only a " post - hoc " retraining .
third , it may underestimate adherence to hand hygiene because it does not consider standard handwashing , which is an effective procedure actually used by many health - care workers ( since it is perceived as more secure ) , even if not the currently recommended standard .
in conclusion , our experience provides a new , reliable method for measuring hand hygiene adherence that noninfection control experts may find easier to understand , compared to conventional ones .
this is a key issue , since no satisfactory infection control can be obtained without the health - care workers collaboration and adherence to hygiene interventions . | summaryintroduction.hand decontamination with alcohol - based antiseptic agents is considered the best practise to reduce healthcare associated infections .
we present a new method to monitor hand hygiene , introduced in a tertiary care pediatric hospital in northern italy , which estimates the mean number of daily hand decontamination procedures performed per patient.methods.the total amount of isopropyl alcohol and chlorhexidine solution supplied in a trimester to each hospital ward was put in relation with the number of hospitalization days , and expressed as litres/1000 hospitalization - days ( world health organization standard method ) . moreover , the ratio between the total volume of hand hygiene products supplied and the effective amount of hand disinfection product needed for a correct procedure was calculated .
then , this number was divided by 90 ( days in a quarter ) and then by the mean number of bed active in each day in a unit , resulting in the mean estimated number of hand hygiene procedures per patient per day ( new method).results.the two methods had similar performance for estimating the adherence to correct hand disinfection procedures .
the new method identified wards and/or periods with high or low adherence to the procedure and indicated where to perform interventions and their effectiveness .
the new method could result easy - to understand also for non - infection control experts.conclusions.this method can help non - infection control experts to understand adherence to correct hand - hygiene procedures and improve quality standards . |
four decades after its introduction for joint arthroplasties , uhmwpe still represents the gold standard as an articulating counterface for arthroplasties , since it combines superior wear resistance along with high fracture toughness and biocompatibility compared to other polymers .
it has been demonstrated that the main factor responsible for the failure of uhmwpe in joint replacements is oxidative degradation , which decreases its mechanical properties [ 13 ] .
decreased abrasive wear resistance , due to oxidation , leads to the formation of wear debris and consequently to osteolysis , which has been recognized as being the main cause of failure in orthopaedic implants [ 14 ] .
the mechanical properties are of great relevance when the uhmwpe inserts are subjected to high contact stresses that can exceed the yield stress of the uhmwpe , leading to permanent deformation and to the catastrophic rupture and failure of the implant .
oxidation is strictly correlated with the sterilization method : uhmwpe components sterilized with ethylene oxide ( eto ) do not oxidize , while those sterilized using high - energy radiation in air ( radiation or an electron beam with a dose of 2540 kgy ) are known to show high levels of oxidation [ 3 , 69 ] .
immediate oxidation following the high - energy treatment results in chain scission in the uhmwpe , thus immediately decreasing its molecular mass and therefore its mechanical properties .
moreover , the oxidative degradation proceeds during storage and in vivo , thus further exacerbating the problem [ 810 ] .
several methods have been applied to reduce the impact of the oxidation on the duration of the implant : thicker inserts have been recommended , the designs of the implants have been improved , and the use of ionizing radiations in air for sterilization has been avoided . in 1998 , in the safety notice 98161998 ( uhmwpe components of joint replacement implants ) , the british medical devices agency established that uhmwpe components should not be used if they are over five years old and if they have passed their indicated expiry date . in 2005 , the italian ministero della salute ( see dgfdm / iii/7101/p / i.1.c.r .
8/3/2005 ) recommended that the use of implants sterilized in the presence of oxygen should be avoided .
recently , radiation crosslinking of the uhmwpe has been applied as answer to the main problem , wear debris related osteolysis .
alternative bearing materials , such as ceramics and metals , are associated with concerns about biocompatibility , duration , carcinogenicity , revision difficulties and costs , which explains why joint replacements using polyethylene inserts are still the ones mostly commonly used in orthopaedics .
this review discusses the historical issues associated with polyethylene , such as oxidation , sterilization method and storage , as well as
the final aim is to aid the orthopaedic surgeons in the selection of the implant and polyethylene insert , the information given to the patient , and when signing contracts .
medical - grade polyethylene ( uhmwpe with an average molecular mass of > 2,000,000 a.m.u . ) is a semicrystalline polymer that can be depicted as a set of ordered regions ( crystalline lamellae ) embedded in a disordered amorphous phase .
the degree of crystallinity is an important parameter : higher crystallinity gives a larger modulus of elasticity , superior yield strength , improved resistance to creep deformation and enhanced fatigue strength , all of which are desirable properties for joint components .
the degree of crystallinity , within the range commonly used for medical - grade uhmwpe , does not substantially affect the wear resistance , which is related to the molecular mass .
the resistance to creep deformation of the uhmwpe is important to evaluations of the relative contribution of deformation or wear to the penetration of the femoral head into the insert .
the fatigue strength is also very important , since it relates to the ability of uhmwpe to resist cyclic damage modes , which are very common in knee components and also in hip components , although prevalent in the rims of malpositioned cups .
medical - grade uhmwpe orthopaedic implants are machined from stocks and sheets made from uhmwpe powders by compression moulding or ram extrusion and subsequent annealing ; the astm f 648 - 07 designation ( standard specification for uhmwpe powder and fabricated form for surgical implants ) defines the characteristics required for medical - grade orthopaedic uhmwpe : density excluded , there are no upper limits on any of the starting parameters , and the characteristics of the material are determined before processing and sterilization .
it is clear that commercially available uhmwpe inserts can be very different from each other after processing , sterilization and packaging , which is very relevant to their clinical applications .
when a polymeric material is exposed to a stronger energy than that of the chemical bonds , the consequence is bond scission and the formation of free radicals ; this chain fragmentation modifies the mechanical properties of the polymer .
high - energy radiation ( -rays , x - rays and electron beams ) , heat and strong mechanical stress are all examples of energies that can break chemical bonds . even if only a single c c bond of the polymer chain in uhmwpe is broken and twoch2 radicals are formed , the molecular mass decreases ; as a consequence , many of the chemical and physical properties of the polymer begin to worsen . in orthopaedics ,
this issue is mainly associated with the radiation and electron beams commonly used during sterilization , and the process is known as degradation . if oxygen is present when the degradation process occurs , it is called oxidative degradation ( oxidation ) . once the oxidation process ( which is also a function of the temperature ) has been initiated , it can not be interrupted , and its rate increases continuously with a series of reactions that involve free radicals and oxygen .
the extent of the oxidative process depends on the number of radicals formed during sterilization and on the amount of oxygen , which can be either atmospheric , present at the sterilization , or it can be oxygen that penetrated by diffusion into the polymer during processing and storage or while the joint is being used in vivo . therefore , the oxidative degradation can continue during storage and in vivo implantation [ 14 , 15 ] .
sterilization of uhmwpe components deserves a special mention since , as described above , it is known that this process can modify the mechanical and wear properties of uhmwpe [ 1 , 69 , 16 ] .
high - energy radiation represents the most common sterilization technique : the source of radiation is the decay of an unstable co nucleus , while electron beams are generated from the electrons emitted by a thermally excited tungsten filament , which are accelerated by electric fields .
the dose absorbed by the material during sterilization depends on the geometry of the sample and its position in relation to the source .
the electron beam is easier to control and requires a shorter period of treatment ( seconds ) .
uhmwpe components are usually stored on the shelf for long durations prior to implantation ( periods of six months or longer ) ; in addition , uhmwpe inserts of total joint replacements have historically been packaged in air and thereafter sterilized by radiation .
it is well established that such irradiation , as well as electron beam irradiation , causes crosslinking , chain scission and long - term oxidative degradation of polyethylene , and that long - term post - irradiation aging can have detrimental effects on the morphology and mechanical properties of uhmwpe [ 1 , 14 , 16 , 17 ] .
macroscopic evidence for the oxidative degradation caused by -sterilization in air can be seen on a uhmwpe section , where it looks like a white halo and is called the crown effect or white band ; this is a zone where a critical molecular mass decrease has occurred , and which therefore has very low mechanical properties , resulting in the well - known effects of delamination and fracture that are typical of such components .
it was previously erroneously believed that oxidation was associated with fatigue damage mechanisms ; however , it has since been established that there is a correlation between the rate of abrasive wear and the post - oxidative reduction in molecular weight [ 2 , 3 ] . in response to these oxidation issues
, some manufacturers now sterilize uhmwpe using non - radiation - based methods , such as ethylene oxide ( eto ) or ( more recently ) gas plasma sterilization ( gp ) ; sterilization by steam is not feasible because the temperatures required about 135c could result in modifications to the material .
the treatment is continued for as long as needed for the gas to diffuse inside the containers ; the packages are then left under vacuum for enough time to allow the complete elimination of eto .
prosthetic uhmwpe sterilized with eto does not undergo any variation in chemical and physical structure .
gas plasma is a surface sterilization method based on the action of ionized gas ( i.e. , hydrogen peroxide or peracetic acid ) , which deactivates biological organisms . commercially available
gp sterilization methods are usually carried out at low temperatures ( below 50c ) and do not significantly affect the physical , chemical and mechanical properties of uhmwpe
. a detailed mechanism for the oxidation for orthopaedic implants has been described , and it has been demonstrated that oxidation can also occur under certain conditions in ethylene oxide sterilized uhmwpe , albeit to a much smaller extent than for -radiation - sterilized uhmwpe .
however , this phenomenon has been related to the presence in the pristine resin of calcium stearate , which is no longer used in contemporary medical - grade uhmwpe [ 79 ] .
as another response to long - term post - irradiation ageing and oxidation , some manufacturers have recently shifted to sterilization with high - energy radiation performed in vacuum or under inert gases ( nitrogen or argon ) .
the material used for the envelope or packaging itself which can classified into the following categories : ( a ) gas - permeable packaging ; ( b ) polymer barrier packaging , and ; ( c ) aluminium barrier packaging is clearly important .
the gas - permeable packaging used is usually a pet ( polyethylene terephthalate ) blister with a tyvek cover , which allows the diffusion of gases ( oxygen included ) ; it is therefore indicated for eto or gas plasma sterilization , but it does not prevent oxidative degradation when used for radiation sterilization . polymer barrier packaging is based on a series of multilayer plastic bags with gas - barrier properties , and therefore has a limited but measurable permeability to oxygen ; it does not exclude the presence of oxygen during and after the radiation sterilization .
aluminium barrier packaging is virtually impermeable to gases , and so only oxygen already dissolved in the uhmwpe prior to irradiation can be present . a complete absence of sterilization - induced oxidation can only be guaranteed by gas sterilization , particularly because the extent to which in vivo oxidation rates affect the clinical performance of conventional uhmwpe packaged in low - oxygen environments and then sterilized using radiation is still unclear .
abrasion wear is the process of removing parts of a material from the surface during reciprocal movement along another surface with greater hardness . in orthopaedic
joint components , the uhmwpe is removed because the interactions of its chains are weak compared to those between the metal or ceramic atoms in femoral head and femoral knee components . the particles of polyethylene removed induce aseptic loosening , through a mechanism involving the formation of reactive tissue and consequently osteolysis , which has been recognized as being the main cause of implant failure [ 4 , 19 ] .
the exact immune reaction that occurs in periprosthetic osteolysis of joint replacements is still unclear : it is known that several types of immune processes appear to be relevant . a foreign - body , granulomatous response to uhmwpe particles denotes a nonspecific chronic inflammatory reaction involving activated mononucleated macrophages and fibroblasts but few t lymphocytes .
the activation of macrophages has been related to the size , shape , volume and number of radiation - sterilized uhmwpe debris particles : those 0.310 m in size are phagocytable and are therefore the most biologically active [ 21 , 22 ] .
an influence of the chemical composition of the uhmwpe particles has recently been suggested : the reactivity might be related to the composition of the surfaces of the particles themselves ( superficial reactivity ) , and in particular to the level of oxidation of the uhmwpe itself [ 23 , 24 ] .
oxidized particles from -irradiated uhmwpe inserts would be more effective at activating the macrophages than the unoxidized particles from eto - sterilized uhmwpe .
the surface reactivity of the particles also depends on the properties of the absorbed molecules , their hydrophilic / phobic character , and the release of radicals which can react with human tissues .
many modifications can occur : freshly detached particles are different from particles that have been in contact with biological tissues for some time , and the debris may be not only fragments of uhmwpe but fragments of an oxidized , lower molecular mass polyethylene .
actually , catastrophic failures due to extreme wear and heavy oxidation are quite uncommon ; nevertheless , wear is also a function of time , and therefore abrasion and the production of abraded particles remains a problem in young , active patients with long life expectancies .
polymer crosslinking is a well - known process in chemistry : it involves the linking of two or more molecular chains through chemical covalent bonds . amongst the several methods that can be employed to achieve this ,
crosslinking is obtained in orthopaedics by high - energy irradiation , which leads to the formation of radical species that react with chain imperfections and other radicals .
such reactions result in polymer chains with stable c c chemical bonds , theoretically increasing the molecular mass to infinity [ 13 , 25 ] . basically , crosslinked uhmwpe ( xpe ) has much better wear resistance and decreased mechanical properties compared to conventional uhmwpe .
the potential benefits of reduced particulate wear generation led to the introduction of crosslinked uhmwpe in orthopaedics during the late 1970s . following laboratory wear tests that confirmed the theoretical decrease in the wear rate , xpe has been widely used since the late 1990s as a bearing surface for orthopaedic implants [ 28 , 29 ] .
medical - grade crosslinked polyethylenes for orthopaedics are processed with radiation doses of 60100 kgy at different temperatures and are then thermally treated to remove residual radicals .
one of the major advantages of post - irradiation thermal treatment is that it also imparts oxidation resistance to the material , due to the removal of detectable amounts of residual free radicals and hydroperoxides .
nevertheless , only melting is completely effective at eliminating the residual free radicals and the hydroperoxides formed during radiation sterilization , and therefore at preserving uhmwpe from radiation - related oxidative degradation [ 30 , 31 ] .
in contrast , the problem with complete melting is the resulting deterioration in mechanical properties like elongation - to - break , tensile modulus , tensile strength [ 28 , 32 ] and j - integral fracture toughness , and resistance to fatigue crack propagation [ 3337 ] .
uhmwpe melting erases the thermal history induced by ram extrusion and compression moulding ; since cooling or recrystallization after melting are carried out without applying any pressure , the process decreases the overall degree of crystallinity of radiation - crosslinked uhmwpe . in theory ,
one possible method of restoring crystallinity in crosslinked uhmwpe would be to utilize high - pressure crystallization , but this is not possible using current processing technology .
several new methods are now currently used in order to resolve the crystallization issue and to impart higher mechanical properties to the crosslinked polyethylene , like annealing close to but below the melting temperature of crosslinked uhmwpe , solid - state deformation followed by annealing , and repetitive subsequent annealings .
however , detectable levels of free radicals still persist in the material , and so it still has a lower resistance to oxidation than remelted crosslinked uhmwpe ; this is undesirable , since it would expose the material to degradative oxidation , which can also be very effective at causing dramatic insert failures for crosslinked materials . in conclusion
, crosslinked polyethylenes can have very different mechanical properties due to the different crosslinking processes that are possible , while uhmwpe , if processed and sterilized correctly , always has the mechanical property values required by the astm . despite the variability
, it should be noted that crosslinked uhmwpe inserts have better mechanical properties than the standard required .
the mechanical properties , and the resistance to fatigue crack propagation in particular , are important in joint component applications , and so it is still unclear whether the benefits of wear resistance due to crosslinking would outweigh the risk of fatigue failure over the long term ; to our knowledge , there are no reports of fatigue failures of crosslinked implants .
it has been demonstrated that wear particles generated by crosslinked polyethylenes play a different role in biological reactions than those generated by conventional polymers , although why this is so is not clearly understood .
crosslinking would generate a larger percentage of small particles [ 39 , 40 ] , which would lead to a higher release of tumor necrosis factor - alpha and therefore to a higher reactivity .
however , crosslinked and conventional polyethylenes would cause similar levels of cytokines , il-6 , il-1 , il-1 and tnf- , and the degree of crosslinking - related osteolysis would be reduced compared to conventional osteolysis .
regardless , radiation - crosslinked uhmwpe acetabular cups and tibial plateaus are now in clinical use , and it is still to be determined whether they will lead to a higher survivorship over the long term compared to conventional noncrosslinked uhmwpe , as expected .
another advantage of using crosslinked polyethylene is the possibility , thanks to its resistance to abrasion , of reducing the thickness of the insert and consequently using larger femoral heads in tha , which reduce the dislocation rate and improve range of motion .
in contrast , fractures of the superior rim of the cup have been correlated to excessive thinness of the polyethylene .
actually , assessments of the clinical behaviour of crosslinked uhmwpe depend on radiographic measurement analyses : these demonstrate an initial penetration of the femoral head into the crosslinked insert , followed by a decreased penetration after the first year compared to conventional inserts .
the initial penetration observed in crosslinked inserts , in vivo and not in laboratory wear tests , has been explained as creep deformation and not as wear .
the results indicate decreased wear for crosslinked uhmwpe compared to conventional uhmwpe . to our knowledge
, most studies have reported good crosslinked insert performance regardless of the manufacturer [ 11 , 4651 ] ; moreover , there is no evidence of large - scale failures of knee or hip implants with crosslinked components due to particle - induced osteolysis , only sporadic case reports of fatigue failure [ 45 , 53 ] .
surface cracking , abrasion , scratching and pitting have been reported on the articular surfaces of retrieved crosslinked acetabular liners , and these features have been explained as being due to the decreased ductility and fatigue resistance associated with extensive crosslinking . in contrast , some studies state that the abovementioned microscopic damage to the surfaces of retrieved crosslinked inserts is a sign of load - induced plastic deformation of the surface , not an early sign of a future failure [ 5457 ] . one final unclear issue regarding crosslinked polyethylene
is its third - body wear resistance : it has been supposed that xpe , due to its micromorphology , could be less resistant to such wear than uhmwpe , which may be a problem when third bodies like bone fragments are present in revisions or microparticles of pmma are present in cemented implants .
nevertheless , to our knowledge , there are no laboratory wear tests that report such a problem , and there are no reports of failures related to the presence of third bodies , so more studies are needed to clearly understand this issue .
in the near future , we can expect radiation crosslinking processes to be optimized to improve the resistance to particulate wear without significantly decreasing mechanical properties ; these developments are of particular importance for knee arthroplasties , where high cyclic stresses can lead to fatigue wear mechanisms . despite the several thermal treatments proposed , as discussed above
, oxidation can be a problem for crosslinked uhmwpe inserts , although to a degree that depends on the processing procedure [ 38 , 49 ] .
in several applications , such as food packaging and preservation , polyethylene is currently stabilized against oxidative degradation by adding a suitable biocompatible stabilizer : vitamin e or ( better ) its synthetic derivative , alpha - tocopherol [ 59 , 60 ] .
therefore , in order to combat oxidation in irradiated crosslinked uhmwpe , the use of a biocompatible and nontoxic antioxidant such as vitamin e has also been proposed .
this would lead to the double advantage of preventing the long - term oxidation associated with the presence of free radicals and preserving mechanical properties [ 6265 ] ; nevertheless , the use of any additive , including antioxidants , in medical - grade uhmwpe ( astm f648 ) is prohibited , which has hindered the use of vitamin e in joint replacements for a long time .
a new standard related to the conditions required for the addition of vitamin e has been approved ( astm standard f 2695 2007 : standard specification for ultra - high molecular weight polyethylene powder blended with alpha - tocopherol [ vitamin e ] and fabricated forms for surgical implant applications , astm international , west conshohocken , pa , usa , see http://www.astm.org ) , even though the old regulation has not yet been cancelled .
recently , given the efficacy of alpha - tocopherol in stabilizing uhmwpe against oxidative degradation , and due to its proven biocompatibility , vitamin e has been introduced at an experimental level by the fda ( regulation 510k ) in order to develop orthopaedic implants that are resistant to oxidation [ 60 , 66 ] .
many manufacturers are now developing crosslinked uhmwpe inserts containing vitamin e ; however , some new concerns have arisen , in particular about the method by which the antioxidant is introduced into the polymer : it can be added before the irradiation [ 63 , 67 ] , during moulding or extrusion ; or by diffusion after irradiation .
the disadvantages of these two methods are that , in the former , crosslinking is suppressed to a minor degree during irradiation , and in the latter , it is difficult to control the concentration and the distribution of the antioxidant . in both cases , the hypothesized advantage is that the vitamin e protects the crosslinked polyethylene against oxidation [ 68 , 69 ] . at the moment ,
the elimination of post - irradiation melting in order to optimize the mechanical properties is just a fascinating hypothesis .
however , it must be noted that the use of vitamin e does not completely suppress oxidation during sterilization with high - energy radiation ; it only retards the process .
it should also be underlined that , even though the safety and biocompatibility of vitamin e is well known , this is still an additive with no clinical history in joint replacement components .
uhmwpe liners can serve well as bearing surfaces for joint replacements . in particular , if the sterilization and packaging processes are carried out correctly , the material has tribological and mechanical properties that can ensure long in vivo service as an articulation , greatly reduced wear , and particle biocompatibility , all without causing catastrophic ruptures and tissue reactions .
in fact , to our knowledge , there are no reports of failures related to the mechanical properties of components made of eto - sterilized uhmwpe and used for arthroplasties .
since the processing techniques play a fundamental role in the durability of implants , the manufacturer could be considered to be the main agent responsible implant durability .
for the same reasons , orthopaedic surgeons must pay careful attention to the processes to which the insert have been subjected : for example , and electron beam irradiation can further produce oxidation , even when conducted in the absence of oxygen
. the full processing history ( sterilization , packaging , time of storage ) of the implant , an indication of its integrity , must be present by law on the labels accompanying it , and must be considered when selecting the joint prosthesis , both before and during surgery .
in fact , in cases where the surgeon is called upon to explain his choice of implant , a complete knowledge of the materials involved and the reasons for choosing the particular implant selected can be helpful .
new and promising materials , like crosslinked and vitamin e charged polyethylenes , are now considered safe but innovative and are therefore handled cautiously : many in vitro tests and several in vivo demonstrations have confirmed the validity of these materials , but it is important to remember that they do not yet have long - term clinical histories .
open issues include : the role of debris of crosslinked polyethylene , the quantity and reactivity of which are still to be elucidated , the long - term behaviour of crosslinked material under the kinds of mechanical stresses encountered in knee arthroplasties , and the interaction of vitamin e with the surrounding tissues as well as its long - term effects on crosslinked and noncrosslinked polyethylene . in conclusion
, surgeons can use the innovative and promising products available on the market , but they should also be aware that some products have been tested over the long term and are safe for clinical use while others have short clinical histories and require caution . | wear debris related osteolysis is recognised as being the main cause of failure in joint replacements based on uhmwpe inserts .
however , many solutions and new polyethylenes have been suggested in order to address this issue .
this review discusses historical issues associated with uhmwpe , such as oxidation , sterilization method and storage , as well as
new
topics , such as crosslinking and stabilization .
the final aim is to aid orthopaedic surgeons in their selection of polyethylene inserts and in the information given to the patients .
the main problem for the polymer is degradative oxidation , which is caused by the combination of the irradiation used for sterilization and oxygen , and which leads to a decrease in wear resistance and mechanical properties .
irradiation and packaging in the absence of oxygen can only reduce the oxidation , while sterilization with gas ( eto or gas plasma ) is the only method that effectively eliminates it .
manufacturing processes are of great relevance to the clinical duration and must be considered by surgeons .
crosslinked polyethylene has been developed for joint inserts due to its superior wear resistance compared to conventional uhmwpe ; to prevent the oxidation , crosslinked polyethylene requires post - irradiation thermal treatment , which reduces its mechanical properties and which depends on the producer .
several good clinical results from the use of crosslinked acetabular cups have reported at mid - term , while early results for knee replacements are also encouraging .
recently , the use of the antioxidant vitamin e ( alpha - tocopherol ) has been introduced for joint prostheses in order to prevent the oxidation of both crosslinked and noncrosslinked uhmwpe . |
he had a 5-year history of schizophrenia and was previously partially controlled on risperidone 6 mg with trihexyphenidyl 4 mg in two equally divided doses daily in the last 2 years .
then he was switched to amisulpride 200 mg twice daily , that initially controlled the akathisia and agitation symptoms . however , this dose of amisulpride ( 400 mg in two divided doses ) for 2 months did not achieve full symptom remission although he was better than before with akathisia remitted and psychomotor agitation controlled but still had disorganized behavior , fleeting delusion and hallucination . in an attempt to achieve complete symptom remission
the patient was switched to amisulpride 200 mg thrice daily ( 600 mg / day ) for next 6 weeks .
unfortunately , the patient did not achieve any further symptom remission , but did not deteriorate either . at this juncture ,
however , as the patient has already partially responded to amisulpride without much side - effect and amisulpride augmentation of clozapine is a proven effective procedure , to achieve further clinical improvement this augmentation strategy were thought of . here
reverse augmentation strategy i.e. , to augment amisulpride with clozapine ( 25 mg to start with , which then escalated to 75 mg with 25 mg increments every 3 days ) was tried , with an expectation of achieving symptom remission , as these two medicines may have synergistic effect . however , within 2 weeks this patient presented severe akathisia , restlessness , hypertonia along with sialorrhoea and tachycardia .
these were managed with propranolol 120 mg and trihexyphenidyl 4 mg , but with modest success for 1 week .
as clozapine was the medication last introduced , it was withdrawn and the patient continued the other medications , i.e. , amisulpride 600 mg / day in three divided doses along with propranolol 120 mg and trihexyphenidyl 4 mg .
moderate improvement of akathisia and agitation symptoms was noted after 1 month , but it did not reach the base level , which were present before start of clozapine .
sialorrhoea was completely subsided on this regime . in order to achieve total side effect remission ,
amisulpride was completely withdrawn under the cover of lorazepam 6 mg and propranolol 120 mg . in another 2 weeks , akathisia symptoms drastically remitted but psychotic symptoms like aggressiveness , disorganized speech and behavior , use of abusive language severely increased .
it is known that clozapine has one of the least tendencies to cause akathisia symptoms on its own and withdrawal of clozapine did not remit akathisia and agitation completely , led to a hypothesis that it may not be clozapine per se , but its interaction with amisulpride might be instrumental in generation of the akathisia symptoms .
so it was decided to give clozapine as a mono therapy a try , as it is a proven effective agent in treatment resistant schizophrenia .
hence clozapine was reintroduced without amisulpride at 50 mg / day , which was increased to 150 mg / day with small increments of 25 mg weekly .
this regime showed very significant symptom control without any akathisia , along with propranolol 120 mg and lorazepam 4 mg .
even after slow withdrawal of supportive medications like propranolol and lorazepam the patient maintained effective symptom control without any akathisia or other unwanted side effect .
only one case report and another case in a case series report have reported positive results attempting clozapine augmentation of patients after non successful treatment of amisulpride . however , this case reported here have shown an occurrence of unwanted side effects in the form of akathisia , dystonia and sialorrhoea after addition of clozapine . whereas , sialorrhoea can solely be due to clozapine , which was subsided after withdrawal of clozapine , akathisia , and dystonia is very unlikely to be caused by clozapine at a dose of 75 mg / day .
it is rather more likely to be caused by amisulpride , especially after an escalation of dose . here
amisulpride dose was not escalated but side effects occurred , which was time related to addition of clozapine . though amisulpride serum level could not be measured in this case , from clinical setting , it was very evident that most probably there may be increase in amisulpride serum level due to drug interaction with clozapine .
it is known that amisulpride is only sparingly metabolized by liver enzymes and thus it is not known to participate in many drug interaction .
however , an article had already reported that there was increase of blood level of amisulpride on addition of clozapine .
this interaction might be a probable explanation for this case 's adverse effect . as the symptoms persisted even after complete withdrawal of clozapine and administration of supportive medications in proper doses
. it also raises the question , whether elevation of amisulpride blood level is instrumental in obtaining good result in augmentation strategy , as a study has already shown that amisulpride does nt seem to increase the blood level of clozapine .
interestingly these two pharmacokinetic observations can not fully explain the reason , why patients resistant to both the medication alone , can respond to their combination therapy
but question may be raised whether sequence of introduction of medications may have some role in this augmentation strategy , as clearly it is safer to augment a partial responder with optimal dose of clozapine with amisulpride than the reverse , because there are more chances of producing side effects in the later schedule .
further systematic research is required to study , whether it is mere association or sequence of introduction of medications may have some role in augmentation strategies and also the exact nature of amisulpride , clozapine drug interaction and possible reasons behind it . | amisulpride is a newer antipsychotic , which is very effective on its own , as well as augmenting other antipsychotic clozapine , which is an effective molecule for treatment resistant schizophrenia . in most cases ,
amisulpride is added on , in partial responders to clozapine . here a case is reported where clozapine was added on , in an amisulpride partial responder but this produced side effect and had to be discontinued .
the case later responded to clozapine alone .
it has been discussed about possible reasons of this finding .
it has also been suggested if sequence of introduction of medication is critical regarding getting the desired effect of the augmentation strategy . |
bioconjugation of both the anti - icam-1 antibody and rat igg ( sigma , st .
louis , mo ) to diethylenetriamine pentaacetic acid bisanhydride ( dtpaba ) and gadolinium chloride ( gdcl ) was performed according to a previously described procedure ( 19 ) .
briefly , the anti - icam-1 antibody was purified from the culture supernatant of rat hybridoma using protein a / g - coupled affinity chromatography .
the anti - icam-1 antibody and rat igg ( control ) were conjugated to dtpaba in pbs ( ph 8.5 ) for 1 hour at room temperature .
gdcl was dissolved in deionized water and added to the dtpa - antibody conjugate solution .
one part of the dtpa - antibody complex was then combined with 40 parts of gdcl in 0.5 m sodium acetate ( ph 5.5 ) .
the molar ratio of gd to antibody was about 1:20 in quantitative analysis using icp - ms ( agilent 7500a , palo alto , ca ) .
the gd - conjugated antibodies did not show any cytotoxic effect on the cultured endothelial cells .
the calculated plasma half - life of the gd - dtpa - antibody conjugate was 3.6 0.16 hours , which is , in turn , ten times longer than that of gd - dtpa ( ~20 min ) .
the measured relaxivity ( mmol-1sec-1 ) of the gd - dtpa - anti - icam-1 ( 23.27 1.31 ) was higher than that of gd - dtpa ( 6.55 0.58 ) as measured on a 1.5 tesla mr scanner .
male dba/1j mice , 7 - 9 weeks of age and free of murine - specific pathogens , were purchased from orient ( seoul , korea ) , housed throughout the experiments in a laminar flow cabinet . to induce cia ,
mice were immunized intradermally at the base of the tail with 100 g of bovine type ii collagen ( cii ; chondrex , redmond , wa ) emulsified in an equal volume of complete freund adjuvant ( chondrex ) .
the day of the first injection of cii was considered day 0 for all mice .
all animal procedures were conducted with approval from our institutional animal care and use committee .
we used three groups of mice : normal non - arthritic mice ( n = 3 ) ; mice in the early inflammatory phase of cia ( day 28 , 4 weeks after 1st injection , n = 9 ) ; and mice in the chronic destructive phase of cia ( day 56 , 8 weeks after 1st injection , n = 3 ) . prior to all mr imaging , all experimental mice were sedated by an intramuscular injection of 10 mg / kg of ketamine hydrochloride ( ketalar ; yuhan yanghang , seoul , korea ) in combination with 10 mg / kg of xylazine hydrochloride ( rumpun ; bayer korea , seoul , korea ) . for mr imaging ,
t2-weighted fat - suppressed images were obtained before injecting contrast agents to evaluate the presence or absence of synovitis in the individual knee joints in all experimental mice .
a t1-weighted dynamic mr imaging was then performed prior to and immediately after , as well as 10 minutes , 30 minutes , 80 minutes , 2 hours , and 24 hours after bolus injection of contrast agents into the tail vein .
all nine experimental mice belonging to the early inflammatory phase of cia group were analyzed , with three mice each receiving one of three types of mr contrast agents as follows : gd - dtpa ( 100 nmoles of gd / gm of body weight ) as a non - targeted contrast agent , gd - dtpa - anti - icam-1 ( 10 nmoles of gd / gm of body weight ) as a targeted contrast agent , and gd - dtpa - igg ( 10 nmoles of gd / gm of body weight ) as a control contrast agent .
a t1-weighted dynamic mri using gd - dtpa - anti - icam-1 was also performed in the normal non - arthritic and chronic destructive phase of the cia groups .
in addition , plain radiographs were obtained to assess joint destruction using a full - field digital mammography system . following the mri ,
the mice were sacrificed by cervical dislocation to collect the knee joints for histopathologic and immunohistochemical analysis .
after sedation , the mice were placed in the prone position with their hind legs fixed to a plate with an adhesive bandage .
all the in vivo mr images of cia mice were obtained with a 1.5 tesla clinical mr machine ( magnetom symphony , siemens medical systems , erlangen , germany ) .
to maximize the signal - to - noise ratio , mice were placed with their knees below a small loop coil ( diameter 5 cm ) provided by the manufacturer .
the fat - suppressed , coronal mr images were obtained before and after the intravenous injection of gd - dtpa ( magnevist , bayer healthcare pharmaceuticals , wayne , nj ) , gd - dtpa - igg , and gd - dtpa - anti - icam-1 . imaging protocols for both the pre - contrast and post - contrast mr studies included the following sequences : fat - suppressed t1-weighted fast spin echo ( fse ) ( repetition time msec / echo time msec , 300/14 ; field of view , 42 56 mm ; matrix dimension , 256 154 ) , and fat - suppressed t2-weighted fse ( 1700/103 ; echo train length , 15 ; field of view , 42 56 mm ; matrix dimension , 256 154 ) .
an mri was performed in the sagittal planes for all sequences . for the quantitative analysis , signal intensity
was measured on serial mr images using a region of interest ( roi ) that was identified by two radiologists blinded to the grouping of the mice .
one circular roi with a diameter between 2 and 5 mm was placed in the same location of knee joints , and included high signal intensity areas , which were regarded as the synovial membrane , synovial fluid , and joints capsules .
after the hind limbs were removed , the legs were fixed in formalin for radiographic imaging .
the plain radiographs of the knee joints were obtained using a mammographic imager based on a direct detection flat panel array design ( mammomat novationdr , siemens medical solutions , erlangen , germany ) .
a full field flat panel digital detector measuring 24 cm 29 cm ( maximum matrix size , 3328 4096 ; pixel size , 70 m ) was used .
all images were obtained using exposure settings of 30 kvp and 90 mas and taken with a 1.5x magnification .
the animals were sacrificed , and the knees were taken to be fixed in 10% neutral buffered formalin overnight and decalcified with calci - clear rapid ( diagnostics , atlanta , ga ) overnight .
the knees were embedded with paraffin , and the tissues were sliced into 5 m sections for hematoxylin and eosin ( h & e ) as well as immunohistochemical staining .
anti - icam-1 polyclonal antibody ( bd biosciences , san jose , ca ) was used for immunohistochemical staining .
bioconjugation of both the anti - icam-1 antibody and rat igg ( sigma , st .
louis , mo ) to diethylenetriamine pentaacetic acid bisanhydride ( dtpaba ) and gadolinium chloride ( gdcl ) was performed according to a previously described procedure ( 19 ) .
briefly , the anti - icam-1 antibody was purified from the culture supernatant of rat hybridoma using protein a / g - coupled affinity chromatography .
the anti - icam-1 antibody and rat igg ( control ) were conjugated to dtpaba in pbs ( ph 8.5 ) for 1 hour at room temperature .
gdcl was dissolved in deionized water and added to the dtpa - antibody conjugate solution .
one part of the dtpa - antibody complex was then combined with 40 parts of gdcl in 0.5 m sodium acetate ( ph 5.5 ) .
the molar ratio of gd to antibody was about 1:20 in quantitative analysis using icp - ms ( agilent 7500a , palo alto , ca ) .
the gd - conjugated antibodies did not show any cytotoxic effect on the cultured endothelial cells .
the calculated plasma half - life of the gd - dtpa - antibody conjugate was 3.6 0.16 hours , which is , in turn , ten times longer than that of gd - dtpa ( ~20 min ) .
the measured relaxivity ( mmol-1sec-1 ) of the gd - dtpa - anti - icam-1 ( 23.27 1.31 ) was higher than that of gd - dtpa ( 6.55 0.58 ) as measured on a 1.5 tesla mr scanner .
male dba/1j mice , 7 - 9 weeks of age and free of murine - specific pathogens , were purchased from orient ( seoul , korea ) , housed throughout the experiments in a laminar flow cabinet . to induce cia ,
mice were immunized intradermally at the base of the tail with 100 g of bovine type ii collagen ( cii ; chondrex , redmond , wa ) emulsified in an equal volume of complete freund adjuvant ( chondrex ) .
the day of the first injection of cii was considered day 0 for all mice .
all animal procedures were conducted with approval from our institutional animal care and use committee .
we used three groups of mice : normal non - arthritic mice ( n = 3 ) ; mice in the early inflammatory phase of cia ( day 28 , 4 weeks after 1st injection , n = 9 ) ; and mice in the chronic destructive phase of cia ( day 56 , 8 weeks after 1st injection , n = 3 ) . prior to all mr imaging ,
all experimental mice were sedated by an intramuscular injection of 10 mg / kg of ketamine hydrochloride ( ketalar ; yuhan yanghang , seoul , korea ) in combination with 10 mg / kg of xylazine hydrochloride ( rumpun ; bayer korea , seoul , korea ) . for mr imaging ,
t2-weighted fat - suppressed images were obtained before injecting contrast agents to evaluate the presence or absence of synovitis in the individual knee joints in all experimental mice .
a t1-weighted dynamic mr imaging was then performed prior to and immediately after , as well as 10 minutes , 30 minutes , 80 minutes , 2 hours , and 24 hours after bolus injection of contrast agents into the tail vein .
all nine experimental mice belonging to the early inflammatory phase of cia group were analyzed , with three mice each receiving one of three types of mr contrast agents as follows : gd - dtpa ( 100 nmoles of gd / gm of body weight ) as a non - targeted contrast agent , gd - dtpa - anti - icam-1 ( 10 nmoles of gd / gm of body weight ) as a targeted contrast agent , and gd - dtpa - igg ( 10 nmoles of gd / gm of body weight ) as a control contrast agent .
a t1-weighted dynamic mri using gd - dtpa - anti - icam-1 was also performed in the normal non - arthritic and chronic destructive phase of the cia groups .
in addition , plain radiographs were obtained to assess joint destruction using a full - field digital mammography system . following the mri ,
the mice were sacrificed by cervical dislocation to collect the knee joints for histopathologic and immunohistochemical analysis .
after sedation , the mice were placed in the prone position with their hind legs fixed to a plate with an adhesive bandage .
all the in vivo mr images of cia mice were obtained with a 1.5 tesla clinical mr machine ( magnetom symphony , siemens medical systems , erlangen , germany ) . to maximize the signal - to - noise ratio ,
mice were placed with their knees below a small loop coil ( diameter 5 cm ) provided by the manufacturer .
the fat - suppressed , coronal mr images were obtained before and after the intravenous injection of gd - dtpa ( magnevist , bayer healthcare pharmaceuticals , wayne , nj ) , gd - dtpa - igg , and gd - dtpa - anti - icam-1 .
imaging protocols for both the pre - contrast and post - contrast mr studies included the following sequences : fat - suppressed t1-weighted fast spin echo ( fse ) ( repetition time msec / echo time msec , 300/14 ; field of view , 42 56 mm ; matrix dimension , 256 154 ) , and fat - suppressed t2-weighted fse ( 1700/103 ; echo train length , 15 ; field of view , 42 56 mm ; matrix dimension , 256 154 ) .
an mri was performed in the sagittal planes for all sequences . for the quantitative analysis , signal intensity
was measured on serial mr images using a region of interest ( roi ) that was identified by two radiologists blinded to the grouping of the mice .
one circular roi with a diameter between 2 and 5 mm was placed in the same location of knee joints , and included high signal intensity areas , which were regarded as the synovial membrane , synovial fluid , and joints capsules .
after the hind limbs were removed , the legs were fixed in formalin for radiographic imaging .
the plain radiographs of the knee joints were obtained using a mammographic imager based on a direct detection flat panel array design ( mammomat novationdr , siemens medical solutions , erlangen , germany ) .
a full field flat panel digital detector measuring 24 cm 29 cm ( maximum matrix size , 3328 4096 ; pixel size , 70 m ) was used .
all images were obtained using exposure settings of 30 kvp and 90 mas and taken with a 1.5x magnification .
the animals were sacrificed , and the knees were taken to be fixed in 10% neutral buffered formalin overnight and decalcified with calci - clear rapid ( diagnostics , atlanta , ga ) overnight .
the knees were embedded with paraffin , and the tissues were sliced into 5 m sections for hematoxylin and eosin ( h & e ) as well as immunohistochemical staining .
anti - icam-1 polyclonal antibody ( bd biosciences , san jose , ca ) was used for immunohistochemical staining .
the icam-1-targeted molecular mr images using gd - dtpa - anti - icam-1 as a targeted contrast agent were examined in the early inflammatory phase of cia mice ( fig .
1 ) . first , t2-weighted fat - suppressed mr images of arthritic knee joints demonstrated synovial thickening and joint effusion as an area of hyper - intense signal in all mice in the early inflammatory phase of cia .
when pre- and post - enhanced t1-weighted images were compared , the quick t1 enhancement was shown immediately after injection of gd - dtpa as a non - targeted contrast agent , which persisted for only a few minutes , and completely disappeared within 80 minutes in the arthritic knee ( fig .
in contrast , dynamic mr images with gd - dtpa - anti - icam-1 found the synovial enhancement to increase gradually and persist for 24 hours after injecting this icam-1-targeted contrast agent ( fig .
interestingly , although the gd content of the gd - dtpa - anti - icam-1 was almost ten times lower than that of gd - dtpa , a significant t1 enhancement of inflamed synovium was observed in mr images with the gd - dtpa - anti - icam-1 .
however , the dynamic mr images with gd - dtpa - igg , which was a gd - conjugated control antibody with the same amount of gd as the gd - dtpa - anti - icam-1 , showed signal enhancement for only 30 - 60 minutes . moreover , this t1 enhancement disappeared within 24 hours ( fig .
in addition , mr images in figure 1 show well - preserved joints in the arthritic knees in all the early inflammatory phases of cia mice .
figure 2 reflects quite different patterns of the enhancement curves of these three kinds of contrast agents following the quantitative analysis of signal enhancement in the early inflammatory phase of cia mice .
t1 enhancement ( mean standard deviation [ sd ] : 393.0 49.1 , n = 3 ) was demonstrated immediately after injecting gd - dtpa ( 100 nmoles of gd / gm of body weight ) , compared to pre - enhanced images ( 212.3 35.4 ) and decreased t1 enhancement at 80 minutes ( 254.7 20.2 ) in the early inflammatory phase of cia mice . however , gd - dtpa - anti - icam-1 ( 10 nmoles of gd / gm of body weight ) enhanced images display gradually increasing signal enhancement from immediately following injection ( mean sd : 424.3 35.2 , n = 3 ) to 24 hours ( 532.0 11.3 ) after injection of the targeted contrast agent compared to pre - enhance images ( 293 37.6 ) .
moreover , the mr images with gd - dtpa - igg show signal enhancement for only 30 - 60 minutes after injection of contrast agent compared to pre - enhance images ( 307 19.7 ) , which fades away within 24 hours ( 297 7.6 ) .
the gd - dtpa - igg enhancement patterns of all three cia mice , in the early inflammatory phase , were also similar , although the size of the synovial inflammatory area varied .
in addition , the results of the quantitative analysis of signal enhancement were in accordance with the calculated plasma half - life of gd - dtpa - antibody conjugate ( 3.6 0.16 hours ) , which is ten times longer than that of gd - dtpa ( ~20 minutes ) .
icam-1-targeted molecular mr images using gd - dtpa - anti - icam-1 were also examined in the chronic destructive phase of cia mice
. moreover , t2-weighted fat - suppressed mr images of arthritic joints demonstrated synovitis and joint destruction .
however , very little to no signal enhancement was observed in arthritic knees up to 24 hours after injection of gd - dtpa - anti - icam-1 , compared with mice in the early inflammatory phase of cia ( fig .
the patterns of enhancement curves following the quantitative analysis of signal enhancement of all three cia mice in the chronic destructive phase were also similar ( fig .
, no signal enhancement was observed in the t1-weighted mr images of non - arthritic normal knee joints up to 12 hours after injection of gd - dtpa - anti - icam-1 . also , plain film ,
5a ) . the significant signal enhancement following the injection of gd - dtpa - anti - icam-1 was noted in arthritic knee joints of early phase cia mice ( fig .
5b ) . the joint of this early inflammatory phase of cia mice demonstrated the marked inflammatory changes on t2-weighted mr images and marked synovial hyperplasia on h & e staining .
the evidence of bone destruction was not observed on plain radiograph and h & e staining .
dense icam-1 expression is shown in the endothelium and is reduced in the subintimal layer of the proliferating synovium of mice in this early inflammatory phase of cia . however
, the icam-1-targeted synovial enhancement was not observed in the chronic destructive phase of cia mice , even though they also showed inflammatory changes on t2-weighted mr images ( fig .
severe pannus invasion and bone destruction were observed on h & e staining and plain radiograph , but little to no icam-1 expression was observed in the synovium of the chronic destructive phase of cia mice .
the icam-1-targeted molecular mr images using gd - dtpa - anti - icam-1 as a targeted contrast agent were examined in the early inflammatory phase of cia mice ( fig .
1 ) . first , t2-weighted fat - suppressed mr images of arthritic knee joints demonstrated synovial thickening and joint effusion as an area of hyper - intense signal in all mice in the early inflammatory phase of cia .
when pre- and post - enhanced t1-weighted images were compared , the quick t1 enhancement was shown immediately after injection of gd - dtpa as a non - targeted contrast agent , which persisted for only a few minutes , and completely disappeared within 80 minutes in the arthritic knee ( fig .
in contrast , dynamic mr images with gd - dtpa - anti - icam-1 found the synovial enhancement to increase gradually and persist for 24 hours after injecting this icam-1-targeted contrast agent ( fig .
interestingly , although the gd content of the gd - dtpa - anti - icam-1 was almost ten times lower than that of gd - dtpa , a significant t1 enhancement of inflamed synovium was observed in mr images with the gd - dtpa - anti - icam-1 .
however , the dynamic mr images with gd - dtpa - igg , which was a gd - conjugated control antibody with the same amount of gd as the gd - dtpa - anti - icam-1 , showed signal enhancement for only 30 - 60 minutes . moreover , this t1 enhancement disappeared within 24 hours ( fig .
in addition , mr images in figure 1 show well - preserved joints in the arthritic knees in all the early inflammatory phases of cia mice .
figure 2 reflects quite different patterns of the enhancement curves of these three kinds of contrast agents following the quantitative analysis of signal enhancement in the early inflammatory phase of cia mice .
t1 enhancement ( mean standard deviation [ sd ] : 393.0 49.1 , n = 3 ) was demonstrated immediately after injecting gd - dtpa ( 100 nmoles of gd / gm of body weight ) , compared to pre - enhanced images ( 212.3 35.4 ) and decreased t1 enhancement at 80 minutes ( 254.7 20.2 ) in the early inflammatory phase of cia mice . however , gd - dtpa - anti - icam-1 ( 10 nmoles of gd / gm of body weight ) enhanced images display gradually increasing signal enhancement from immediately following injection ( mean sd : 424.3 35.2 , n = 3 ) to 24 hours ( 532.0 11.3 ) after injection of the targeted contrast agent compared to pre - enhance images ( 293 37.6 ) .
moreover , the mr images with gd - dtpa - igg show signal enhancement for only 30 - 60 minutes after injection of contrast agent compared to pre - enhance images ( 307 19.7 ) , which fades away within 24 hours ( 297 7.6 ) .
the gd - dtpa - igg enhancement patterns of all three cia mice , in the early inflammatory phase , were also similar , although the size of the synovial inflammatory area varied .
in addition , the results of the quantitative analysis of signal enhancement were in accordance with the calculated plasma half - life of gd - dtpa - antibody conjugate ( 3.6 0.16 hours ) , which is ten times longer than that of gd - dtpa ( ~20 minutes ) .
icam-1-targeted molecular mr images using gd - dtpa - anti - icam-1 were also examined in the chronic destructive phase of cia mice . moreover , t2-weighted fat - suppressed mr images of arthritic joints demonstrated synovitis and joint destruction . however , very little to no signal enhancement was observed in arthritic knees up to 24 hours after injection of gd - dtpa - anti - icam-1 , compared with mice in the early inflammatory phase of cia ( fig .
the patterns of enhancement curves following the quantitative analysis of signal enhancement of all three cia mice in the chronic destructive phase were also similar ( fig .
, no signal enhancement was observed in the t1-weighted mr images of non - arthritic normal knee joints up to 12 hours after injection of gd - dtpa - anti - icam-1 . also , plain film ,
the significant signal enhancement following the injection of gd - dtpa - anti - icam-1 was noted in arthritic knee joints of early phase cia mice ( fig .
the joint of this early inflammatory phase of cia mice demonstrated the marked inflammatory changes on t2-weighted mr images and marked synovial hyperplasia on h & e staining .
the evidence of bone destruction was not observed on plain radiograph and h & e staining .
dense icam-1 expression is shown in the endothelium and is reduced in the subintimal layer of the proliferating synovium of mice in this early inflammatory phase of cia . however , the icam-1-targeted synovial enhancement was not observed in the chronic destructive phase of cia mice , even though they also showed inflammatory changes on t2-weighted mr images ( fig .
severe pannus invasion and bone destruction were observed on h & e staining and plain radiograph , but little to no icam-1 expression was observed in the synovium of the chronic destructive phase of cia mice .
we demonstrated that gd - dtpa - anti - icam-1 can be targeted to icam-1 that is expressed in the inflamed synovium of mice in the early inflammatory phase of cia .
notably , this is the first study to identify the utility of targeted molecular mri in the ra research field .
we also showed that molecular mri with gd - dtpa - anti - icam-1 can differentiate the early inflammatory phase of cia from the chronic destructive phase .
our observation shows the possibility of using gd - dtpa - anti - icam-1 as a direct cellular targeting contrast agent in the molecular mri for ra .
monoclonal antibodies have limitations such as an increased circulating half - life and the human anti - mouse antibody response , although they have greater specificity for antigens as molecular imaging agents than conventional non - specific imaging agents .
these shortcomings can produce increased background ( blood pool ) activity and may interfere with biospecificity ( 20 ) . in our study , there are good reasons to conclude that the significant signal enhancement on mr images reflects the targeted icam-1 binding of gd - dtpa - anti - icam-1 rather than both the unbound and the non - specifically bound fractions of this agent .
first , on the mr images with gd - dtpa - anti - icam-1 , the t1 enhancement gradually increased and lasted for 24 hours ( several times longer than the half - life of this icam-1-targeted contrast agent ) in the arthritic joints of cia mice .
in contrast , t1 enhancement was shown immediately following the injection of gd - dtpa as a non - targeted contrast agent , however lasted for only a few minutes . also , the dynamic mr images with gd - dtpa - igg , gd - conjugated control antibody with the same amount of gd as the gd - dtpa - anti - icam-1 demonstrated signal enhancement for only 30 - 60 minutes after the injection of contrast agent . second , although the gd content of the gd - dtpa - anti - icam-1 was almost ten times lower than that of gd - dtpa , a marked amount of t1 enhancement by this icam-1 targeted contrast agent was observed .
third , the signal enhancement by gd - dtpa - anti - icam-1 on mr images and icam-1 expression on synovium , were observed in only the early inflammatory phase of cia mice , but not in the chronic destructive phase .
thus , we concluded that prolonged signal enhancement on our mr images using gd - dtpa - anti - icam-1 is a consequence of targeted binding to icam-1 in inflamed synovium .
gd - dtpa is a non - specific contrast agent that is commonly used for mri of hemodynamic parameters , including blood perfusion and vascular permeability ( 21 ) .
in contrast , gd - dtpa - anti - icam-1 is a specific mr contrast agent that can be targeted to icam-1 and can thus identify specific cellular or molecular events that underlie disease .
gd - dtpa is transported in the plasma and diffuses freely throughout the interstitial space , but it is impermeable to the plasma membrane .
this non - specific contrast agent accentuates differences in permeability or perfusion and is helpful in characterizing physiological processes , such as blood volume , flow , and perfusion ( 21 ) . in ra
, the inflamed synovium shows a large number of capillaries , enhanced capillary perfusion , and permeability .
thus , gd - dtpa allows for the differentiation of the inflamed synovium from both surrounding healthy tissue and normal synovium through a marked increase of signal intensity on t1-weighted mr images ( 22 - 24 ) .
however , this contrast agent is nonspecific and does not provide any information regarding cellular and molecular mechanisms .
importantly , synovitis in ra is characterized by angiogenesis , cellular infiltration , and the production of various cytokines and adhesion molecules . while gd - dtpa can only estimate blood perfusion and vascular permeability , gd - dtpa - anti - icam-1 may enable the non - invasive evaluation of cellular and molecular changes , which often occur before any signs of pathologic and anatomical changes in the course of ra .
to our knowledge , this is the first report that identifies the utility and possible applicability of a targeted molecular mri in the field of research for ra .
moreover , a conventional 1.5 tesla mr machine , which is widely used in clinical practice , was used in our study .
mri has been regarded as a powerful imaging tool because of its noninvasive nature , high spatial resolution , and tomographic capabilities .
in addition , mri can visualize bone erosions much better than conventional radiography in the early stage of ra .
thus , mri has been increasingly used to evaluate the joints of ra patients ( 25 ) .
the development of targeted mr contrast agents could dramatically expand the range of mr applications by combining the noninvasiveness and high spatial resolution of mri with specific localization of molecular targets ( 21 ) .
several successful attempts with various molecular targets and specific mr agents have been reported in the field of tumor imaging ( 26 - 29 ) .
however , mri has not been used for studies using molecular imaging agents in ra , as these studies are currently limited to nuclear and optical imaging methods ( 3 ) .
the full mr imaging examination can be a lengthy procedure , and its low signal sensitivity has been a major limitation in experimental molecular imaging ( 24 ) .
our study used gd - dtpa - anti - icam-1 , which has several unique characteristics including a higher conjugation ratio of gd / antibody ( 1:20 ) , a much longer plasma half - life ( ten times longer than that of gd - dtpa ) , and greater relaxitivity ( two times higher than that of gd - dtpa ) as mentioned in a previous report ( 16 ) .
therefore , a significant amount of t1 enhancement was obtained in the dynamic molecular mri by overcoming the limitations of mri as an experimental molecular imaging method .
limitations of our study include small sample size , no definite discrimination between synovial fluid and targeted synovium , and the absence of an icam-1 ab - blocking study .
thus , further studies are needed to clarify the usefulness of molecular mri using gd - dtpa - anti - icam-1 as guidance for the icam-1 targeted diagnosis and a marker of therapeutic response in clinical practice . in conclusion , this study demonstrates that gd - dtpa - anti - icam-1 can be targeted to icam-1 expressed in the inflamed synovium during the early inflammatory phase of cia mice .
thus , our results clearly show the feasibility of using gd - dtpa - anti - icam-1 as a specific contrast agent for molecular mri that targets icam-1 expressed in the inflamed synovium of cia , the most reliable model of human ra . in ra , this novel tool may be used to evaluate pathophysiological processes , facilitate early diagnosis , and monitor therapy . | objectiveto determine the utility of intercellular adhesion molecule ( icam)-1 antibody - conjugated gadolinium diethylenetriaminepentaacetic acid ( gd - dtpa - anti - icam-1 ) as a targeted contrast agent for the molecular magnetic resonance imaging ( mri ) in collagen - induced arthritis ( cia).materials and methodsthree groups of mice were used : non - arthritic normal , cia mice in both the early inflammatory and chronic destructive phases . the mr images of knee joints were obtained before and after injection of gd - dtpa - anti - icam-1 , gd - dtpa , and gd - dtpa - immunoglobulin g ( ig g ) and were analyzed quantitatively .
the patterns of enhancement on the mr images were compared with the histological and immunohistochemical icam-1 staining.resultsthe images obtained after injection of gd - dtpa - anti - icam-1 displayed gradually increasing signal enhancement from the moment following injection ( mean standard deviation [ sd ] : 424.3 35.2 , n = 3 ) to 24 hours ( 532 11.3 ) , rather than on pre - enhanced images ( 293 37.6 ) in the early inflammatory phase of cia mice . however , signal enhancement by gd - dtpa and gd - dtpa - igg disappeared after 80 minutes and 24 hours , respectively .
in addition , no significant enhancement was seen in the chronic destructive phase of cia mice , even though they also showed inflammatory changes on t2-weighted mr images .
icam-1 expression was demonstrated in the endothelium and proliferating synovium of the early inflammatory phase of cia mice , but not in the chronic destructive phase.conclusionmolecular mri with gd - dtpa - anti - icam-1 displays specific images targeted to icam-1 that is expressed in the inflamed synovium of cia .
this novel tool may be useful for the early diagnosis and differentiation of the various stages of rheumatoid arthritis . |
leishmaniases are diseases with tegumentary or visceral forms that are caused by protozoans of the genus leishmania and are transmitted via insect vectors and affect more than 12 million people in tropical and subtropical areas of the world .
leishmania ( leishmania ) amazonensis is one of the most prevalent species that causes tegumentary leishmaniasis in the new world causing localized cutaneous , mucosal , or diffuse cutaneous leishmaniases .
the control or progression of leishmaniasis in the vertebrate host depends on nonspecific and specific factors of the immune response as well as the ability of the parasite to evade the host response .
however , the various disease manifestations and outcomes may result from diverse leishmania species - related immune and immunopathogenic responses and l. ( l. ) amazonensis exhibits peculiarities that distinguish it from other species .
the differences in the immunopathogenic mechanisms of patients infected with either l. ( viannia ) braziliensis or l. ( l. ) amazonensis , the species that are prevalent in brazil , are evident . while the response to l. ( v. ) braziliensis infection is related to the exacerbation of the th1-type response [ 46 ] or to defects in the modulation of ifn- production or decreased il-10 receptor expression in severe mucosal cases [ 6 , 7 ] , l. ( l. ) amazonensis infection tends to elicit a less effective or limited t cell - response against the parasite [ 5 , 8 ] , which could explain the development of diffuse cutaneous leishmaniasis in certain cases .
these differences are also noticeable in cutaneous leishmaniasis in mice . compared to l. major infection , to which the balb / c mouse strain is susceptible and the c57bl/6 strain is resistant , both mouse strains are resistant to l. ( v. ) braziliensis and both are susceptible to l. ( l. ) amazonensis .
the development of lesions in l. ( l. ) amazonensis - infected mice is not only dependent on the th2 immune response but rather on the presence of th1 cd4 t cells and ifn- , which promotes parasite growth in amastigote - infected macrophages in vitro .
the diverse outcomes and development of specific immune responses during l. ( l. ) amazonensis infection could be attributed to alterations observed during early innate leishmania - host interactions involving inflammatory cytokines , chemokines , toll - like receptors , dendritic cell activation , and so forth .
thus in the present study , we focused on the effect of a growth factor in the nonspecific interaction between l. ( l. ) amazonensis and macrophages . among nonspecific elements ,
we have been studying the effect of insulin - like growth factor ( igf)-i on leishmania and in leishmaniases .
igfs are polypeptides with a molecular mass of approximately 7.5 kda , that exist in two major forms , igf - i and igf - ii , which share 60% similarity and are found in circulation associated with igf binding proteins and in tissues .
igfs affect cell metabolism and are important endocrine growth and differentiation factors . based on these characteristics , we previously studied the effect of igfs on leishmaniasis during their initial interactions as nonspecific factors , though the th1 cytokine ifn- is known to inhibit igf - i , and the th2 cytokines il-4 and il-13 to increase igf - i expression . we have previously shown that adding physiological concentrations of extrinsic igf - i into cultures induces proliferation of different species of leishmania promastigotes and axenic amastigotes , which is an effect that is not observed with igf - ii despite its high similarity to igf - i [ 19 , 20 ] . in experimental models ,
extrinsic igf - i significantly increases lesion sizes and the number of viable parasites . during infection
however , the interaction between amastigotes and host cells is critical for the progression of infection and the establishment of disease , which is different to promastigotes . this fact was elegantly proven recently using axenic leishmania major amastigotes .
specifically , comparing leishmania ( l. ) amazonensis promastigotes and amastigotes showed differences in dendritic cell activation , parasitism after ifn- stimulation , susceptibility to histone proteins , and intracellular signaling after igf - i stimulation .
therefore , in the present study , we analyzed the effects of igf - i on the interaction between leishmania ( l. ) amazonensis amastigotes and macrophages . because our previous data show that igf - i favors parasite growth and arginase activation using leishmania ( l. ) amazonensis promastigotes , we initially evaluated parasitism and arginase activity on amastigote - infected macrophages and on cell - free - amastigotes upon igf - i stimulation . then , we analyzed inflammatory and th1 and th2 cytokine production in infected macrophages upon igf - i stimulation because of their potential role in macrophage activation .
another phenomenon related to the progression of l. ( l. ) amazonensis infection is the phosphatidylserine ( ps ) exposure that was observed only on tissue - derived amastigotes [ 2729 ] .
because no induction factor was identified before and igf - i is present in tissue fluids , we evaluated ps exposure upon igf - i stimulation and the possible role of the ps molecule on macrophage activation . in the present study , we thus show that igf - i induces arginase activity directly in leishmania ( l. ) amazonensis amastigotes and in amastigote - infected macrophages , the latter through the induction of ps exposure on amastigotes that could lead to the alternative activation of macrophages through cytokine - independent mechanisms .
leishmania ( leishmania ) amazonensis ( mhom / br/73/m2269 ) was kindly provided by professor clara lucia barbieri from universidade federal de so paulo and was maintained through regular passage inbalb / c mice .
amastigotes were obtained from footpad lesions of balb / c mice infected one month earlier .
the lesions were aseptically dissected and washed in 0.01 m phosphate - buffered saline ph 7.4 ( pbs ) and cut and ground in a petri dish containing pbs .
then , the suspension was disrupted by four passages through 22-gauge needles and centrifuged 3 times at 250 g for 10 min ; the resulting supernatant was centrifuged at 1,400 g for 30 minutes and the parasites were suspended in rpmi 1640 medium ( gibco , carlsbad , ca , usa ) .
the purified amastigotes were used immediately in the experiments or were expanded to promastigotes and maintained in 199 medium ( gibco ) supplemented with 10% heat - inactivated fetal calf serum ( cultilab , campinas , sp , brazil ) at 26c .
the promastigotes used in these experiments were in the stationary - phase of growth and had undergone no more than four passages in culture .
balb / c mice of both sexes , aged 68 weeks , were obtained from the animal facility of the faculdade de medicina da universidade de so paulo and used as a source of peritoneal macrophages or for the in vivo maintenance of l. ( l. ) amazonensis .
this study was approved by the ethical committee for animal research ( ceea ) , at biomedical sciences institute / universidade de sao paulo , protocol number 041/03 , adhering to the ethical principles in animal research adopted by brazilian college of animal experimentation ( cobea ) .
resident peritoneal cells were obtained from the peritoneal cavities of balb / c mice and suspended in rpmi 1640 medium supplemented with 100 ui / ml penicillin , 100 g / ml streptomycin , and 2% heat - inactivated balb / c mouse serum ( complete medium ) .
the concentration was adjusted to 4 - 5 10 cells / ml and the cells were plated in different amounts either on round 13 mmglass cover slips placed in the wells of 24-well plates ( corning life sciences tewksbury , ma , usa ) or directly into the wells and incubated for 2 h to adhere on the plate at 37c in a humidified atmosphere containing 5% co2 .
thereafter , the wells were washed twice with rpmi 1640 medium to remove any nonadherent cells .
then , either adherent cells ( macrophages ) or leishmania amastigotes or promastigotes were preincubated for 5 minutes with 50 ng / ml recombinant human igf - i or igf - ii ( r & d systems , minneapolis , mn , usa ) and then washed out .
the parasite suspension ( 2 parasites/1 cell ratio ) was dispensed into the wells and allowed to infect for 2 h at 33c in a humidified atmosphere containing 5% co2 and then the noninternalized parasites were washed out and the culture was maintained at 33c in a humidified atmosphere containing 5% co2 . in some cases ,
controls were maintained without igf - i . in some experiments , 100 m n - hydroxy - l - arginine acetate salt ( noha ) ( sigma - aldrich co. , st .
louis , mo , usa ) was added to the culture or used to preincubate the amastigotes for 5 minutes and was washed out .
tests were run in sextuplicate and incubated for 2 , 24 , 48 , or 72 h. the coverslips , supernatants , or cell lysates were recovered for different evaluations . in the experiments for the evaluation of h2o2 production , phenol red free medium was used .
the cover slips were removed from the plates , mounted and stained with giemsa dye and processed for the evaluation of parasitism under a light microscope ( carl zeiss , gttingen , germany ) .
the data are presented as the number of parasites in 100 cells from the formula [ ( number of parasites : number of infected cells ) ( number of infected cells / total number of cells ) 100 ] .
this analysis was performed by two independent observers who were blinded to the experimental conditions .
four million cells in 500 l were plated in 24-well culture plates and infected and stimulated with igf - i as described previously .
some sets were stimulated with ifn- ( 10 ng / ml ) or lipopolysaccharide ( lps ) from escherichia coli ( sigma co. , ltd .
( st louis , mo , usa ) ) ( 1 g / ml ) or noha ( 100 m ) . after 24 h of incubation
, the cells were washed twice with ice - cold pbs and then lysed for 10 minutes with ice - cold lysis buffer ( 10 mm tris - hcl ph 7.6 , 150 mm nacl , 2% np-40 substitute ( octylphenoxypolyethoxyethanol ) , 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride and 5 m leupeptin ) .
the lysed cell preparation was centrifuged at 10,000 g for 5 minutes at 4c and the protein concentration in the supernatant was determined using a lowry protein assay to adjust the concentrations before analysis .
these lysates were used in western blot analyses and arginase expression and activity assays . for the analysis of leishmania amastigote arginase activity ,
cell lysates ( 20 g protein in 20 l ) were run on sodium dodecyl sulphate-10% polyacrylamide gels .
the separated proteins were electrotransferred onto 0.2 mm pore size nitrocellulose membranes using transblot sd - semidry transfer cells ( bio rad , ca , usa ; 30 minutes at 15 mv ) .
the membranes were blocked with 150 mm nacl , 20 mm tris , 0.01% tween 20 , ph 7.4 buffer ( tbs - t buffer ) containing 5% fat - free milk for one hour .
the membranes were reacted with a monoclonal anti - arginase - i antibody ( 1 : 1000 in pbs ) ( bd biosciences pharmigen , san diego , ca , usa ) for 2 h at room temperature , washed three times with tbs - t and incubated with a peroxidase - conjugated polyclonal anti - mouse igg ( 1 : 1000 ) ( sigma - aldrich , saint louis , mo , usa ) for one hour at room temperature . rainbow protein molecular weight markers ( bio - rad laboratories , hercules , ca , usa ) were used . bound antibodies were detected by an ecl chemiluminescence kit ( amersham biosciences , piscataway , nj , usa ) following the manufacturer 's instruction .
cells and amastigotes were taken from the culture and the obtained lysates were assayed for arginase activity as described previously .
briefly , to activate the arginase , 50 l of the lysates were treated with the same volume of 5 mm mncl2 , 25 mm tris - hcl ph 7.4 at 56c for 10 min . then , to 25 l of the activated lysate , 25 l of 0.5 m l - arginine ph 9.7 were added and incubated at 37c for 60 min .
the reaction was stopped with 400 l of h2so4/h3po4/h2o ( 1/3/7 , v / v / v ) .
the urea concentration was measured at 540 nm in a multiskan mcc/340 p version 2.20 plate reader spectrophotometer ( labsystems , vantaa , finland ) after the addition of 25 l of 9% -isonitrosopropiophenone in 100% methanol and incubation at 100c for 45 min .
one unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 mol of urea per minute .
nitrite ( no2 ) accumulation in the cell culture supernatants was used as an indicator of nitric oxide production and it was determined by the griess reaction .
the absorbance of the reaction product at 570 nm was measured using an elisa reader .
the assay is based on the horseradish peroxidase ( hrpo)-mediated oxidation of phenol red by h2o2 as described by pick and keisari .
the cell culture supernatants were harvested after 24 h for the analysis of il-1 , 48 h for il-6 and tnf and 72 h for tgf- and ifn- by elisa according to the manufacturer 's instructions using bd opteia kits ( bd biosciences , usa ) in 96-well tissue culture plates ( corning costar co. , usa ) and read at 450 nm in a multiskan mcc/340 p version 2.20 spectrophotometer ( labsystems , vantaa , finland ) .
the sensitivity of the cytokine assays was as follows : ifn- , 8 pg / ml ; il1- , 8 pg / ml ; il-6 , 16 pg / ml ; tgf- , 20 pg / ml ; and tnf , 8 pg / ml . the concentrations were determined by comparison with a curve generated from each cytokine standard .
all of the cytokine measurements were performed in triplicate and at the same time to avoid intertest variations .
the assay is based on the competitive binding technique in which the pge2 present in the supernatant competes with a fixed amount of alkaline phosphatase - labeled pge2 for sites on mouse monoclonal anti - pge2 antibodies .
the analysis was performed according to the manufacturer 's instructions using a prostaglandin e2 immunoassay ( r&d system , inc .
the detection of annexin v was made by flow cytometry after staining with fluorescein isothiocyanate - conjugated annexin v and propidium iodide ( pi ) using a commercially available kit ( annexin v fitc apoptosis detection kit ii - bd biosciences ) .
amastigotes ( 10 parasite / ml ) were treated in the same way as described above with 50 or 100 ng / ml igf - i or with h2o2 ( 4 or 8 mm ) as a positive control for 24 h. then , the cells were washed twice with cold pbs and 100 l of the solution ( 10cells ) was resuspended in annexin v binding buffer ( 10 mm hepes , 140 mm nacl , 2.5 mm cacl2 ; ph 7.4 ) and incubated with 5 l of annexin v fitc and 5 l of pi at 4c for 15 minutes in the dark .
after this period , 400 l of binding buffer was added to each tube and the cells were collected in a bd facscalibur ( becton dickinson , franklin , nj , usa ) and analyzed by cell quest .
liposomes were prepared by the film - hydration method , using hydrogenated phospholipids ( lipoid gmbh , germany ) . for the preparation of the negatively charged liposomes ( ps - liposomes ) , saturated egg phosphatidylcholine , saturated egg phosphatidylserine , and cholesterol ( sigma - aldrich ) ( 7 : 2 : 1 molar ratio ) or neutral liposomes ( pc - liposomes - control group ) , phosphatidylcholine , and cholesterol ( 9 : 1 ) were dissolved in chloroform : methanol ( 1 : 1 ratio ) .
the mixture was evaporated in a rotary evaporator at 55c at 60 rpm for 40 minutes in a vacuum and protected from light .
a preheated ( 55c ) solution of 2.25% glycerol ( 9 ml ) was added to the lipid film using glass beads .
the swelling process of the preformed liposomes was performed in a rotary evaporator at 55c at 80 rpm for 60 minutes without a vacuum .
the surface charge of neutral and negatively charged liposomes was previously confirmed using the zeta potential analysis , in a zeta plus analyzer ( brookhaven instr .
the samples were diluted in 1 ml of 3 mm kcl as recommended by the manufacturer .
the diluted samples were then analyzed for ten cycles with a voltage of 4 mv .
macrophages ( 2 10/500 l rpmi 1640 medium ) were plated in a 24-well plate and incubated for 2 h to allow adherence at 37c in a humidified atmosphere containing 5% co2 .
thereafter , the wells were washed twice with rpmi 1640 to remove any nonadherent cells .
then , we infected some wells with amastigotes ( 2 parasites/1 cell ratio ) or used 30 l of phosphatidylcholine- ( pc- ) liposome 30% , 30 l of ps - liposome 30% , or 30 l of 2.25% glycerol as stimuli .
we performed the same experiment in parallel using 3 l of an anti - tgf- antibody ( 15 g / ml ) in each well ( r&d systems , inc .
after 24 h , the cells were treated to obtain the lysates and the arginase activity reaction was performed as described above .
the data were analyzed by anova with the student newman - keuls contrast post - test and were considered significant when p < 0.05 . the cytokine absorbances were analyzed after transformation and linear regression .
the kruskal - wallis test with the student newman - keuls contrast post - test was used for statistical comparisons among groups using sigmastat for windows version 3.10 ( systal software , inc . , san jose , ca , usa ) .
using tissue - derived leishmania ( l. ) amazonensis amastigotes , we initially analyzed the parasitism and arginase activity in infected macrophages and extracellular amastigotes .
the effects of igf - i were assayed in the following three different ways : preincubation of amastigotes or macrophages for 5 minutes with igf - i that was washed out or with the factor maintained in the culture medium throughout the experimental period .
we observed an increase in the number of parasites within macrophages after 48 h of amastigote infection in all igf - i stimulation conditions tested ( figure 1(a ) ) , as observed previously with promastigotes .
we did not observe any effect when igf - ii was used with l. ( l ) amazonensis amastigotes ( data not shown ) .
we observed increased arginase expression after igf - i stimulation in infected macrophages ( figure 1(b ) ) . to demonstrate that the enzyme activity was present after igf - i stimulation , we measured the arginase activity by urea determination , and the activity increased after igf - i treatment ( figure 1(c ) ) . to confirm that the urea was derived from arginase activity , a specific arginase inhibitor ( noha )
parasitism increased with arginase activity and was inhibited by noha ( figure 1(d ) ) .
however , nitric oxide production decreased significantly compared to that of controls that lack igf - i ( figure 1(e ) ) .
neither igf - i nor igf - ii altered the production of hydrogen peroxide in l. ( l. ) amazonensis - infected macrophages ( data not shown ) .
furthermore , arginase activity in igf - i - stimulated cell - free - amastigotes was also evaluated and arginase activity increased in these conditions ( figure 1(f ) ) .
the balance between th1 and th2 cytokines is known to be an important determinant in the activation of the l - arginine metabolic pathways that leads to the production of nitric oxide or polyamines and macrophage stimulation with il-4 , il-10 , and tgf- induces arginase - i activation and leads to the increased growth of amastigotes within macrophages [ 30 , 38 ] .
in addition to those cytokines , the production of other inflammatory cytokines ( ifn- , il-1 , il-6 , il-10 , il - l2 , tgf- , tnf , and pge2 ) was analyzed in the leishmania amazonensis amastigote - infected macrophages after igf - i stimulation .
igf - i did not alter cytokine production by amastigote - infected macrophages compared to the controls without igf - i ( figures 2(a ) , 2(c ) , 2(e ) , 2(g ) , and 2(i ) ) . then , as a comparison , we extended the study to analyze cytokine production by leishmania promastigote - infected macrophages upon igf - i stimulation . no alteration in il-10 , il-12 , and pge2
was observed in either leishmania form ( data not shown ) ; however , we observed decreased ifn- ( figure 2(b ) ) but increased tgf- ( figure 2(h ) ) and tnf ( figure 2(j ) ) levels compared with those of the nonstimulated promastigote infected - macrophage controls .
igf - i increased production of il-6 in uninfected macrophages , but while amastigote infection ( figure 2(e ) ) led to its decrease in igf - i - stimulated cells , promastigote infection did not promote il-6 decrease in igf - i - stimulated cells ( figure 2(f ) ) .
il-1 levels remained low in the amastigote - infected cells ( figure 2(a ) ) compared with the high levels in the promastigote - infected cells with or without igf - i stimulation ( figure 2(b ) ) .
because we did not observe effects due to igf - i on cytokine production in the amastigote - infected macrophages , we looked for an alternative mechanism that could explain the alternative macrophages activation , focusing on ps exposure on amastigotes described before .
we investigated the possible effect of igf - i on ps exposure on l. ( l. ) amazonensis after igf - i stimulation .
we used hydrogen peroxide stimulation as a positive control that induced ps exposure on control promastigotes but not on control amastigotes .
however igf - i clearly increased ps exposure on amastigotes ( figure 3 ) but not on promastigotes ( data not shown ) .
we then determined whether ps is involved in inducing arginase activity in macrophages . to restrict the analysis to the effect of this molecule
ps - liposomes have been previously characterized and have exhibited a zeta potential of 88 mv ( 20 ) , which is a considerable negative charge when compared to pc - liposomes , which demonstrated a zeta potential of + 1.25 mv ( 0.2 ) .
neither the pc - liposome nor glycerol induced arginase activity in macrophages ( figure 4 ) . additionally , we observed no change in the arginase activity induced by the ps - liposomes in the macrophages using an anti - tgf- antibody ( figure 4 ) .
in the present study , we used tissue - derived amastigotes to simulate conditions similar to those in infected tissues in vivo .
initially , we observed the alternative activation of macrophages with increased arginase expression and activity and decreased nitric oxide production in l. ( l. ) amazonensis amastigote - infected macrophages , which lead to increased parasitism after igf - i stimulation .
reactive oxygen and reactive nitrogen intermediates are important nonspecific parasiticidal elements and both superoxide and nitric oxide have been previously shown to be necessary for l. ( l. ) amazonensis amastigote killing within macrophages . in our experiments , we evaluated hydrogen peroxide production and found that it was not increased during amastigote or promastigote infection and was not altered upon igf - i stimulation ( data not shown ) .
however , in the present study , only the decrease in no production upon igf - i exposure was sufficient to increase parasitism in the macrophage . regarding this result
, we should consider the additional effect of igf - i on activation of leishmania arginase in our system .
the parasite arginase is vital for its growth and arginase gene deletion has been shown to impair the progression of infection in vitro and in vivo [ 41 , 42 ] .
factors that activate leishmania arginase were unknown , but in our previous study with promastigotes as well as in our present study with amastigotes , we show that a host igf - i does activate parasite arginase . because the observed alternative macrophage activation could have been due to the modulation of cytokine production , we analyzed cytokine production after igf - i stimulus . in l. ( l. )
amazonensis amastigote - infected macrophages , we observed no alteration of cytokines production after igf - i stimulation compared to unstimulated infected controls , suggesting that macrophage arginase activation due to amastigote infection does not involve the modulation of cytokine production .
interestingly , in a parallel analysis , the production of the same cytokines in l. ( l. ) amazonensis promastigote - infected macrophages showed alterations upon igf - i stimulus .
we observed decreased ifn- but increased tgf- and tnf levels compared with those in unstimulated promastigote - infected macrophages , which suggest that increased tgf- and decreased ifn- lead to the alternative activation of macrophages upon igf - i stimulus in l. ( l. ) amazonensis promastigote - infected macrophages .
tgf- production was already increased during infection with l. ( l. ) amazonensis promastigotes as previously observed and was further increased by igf - i exposure in the present study .
the presence of ifn- in the culture supernatant could have originated from contaminant lymphocytes because peritoneal macrophages were used ; however , such lymphocyte contamination , if any , would be very low and the production of ifn- by macrophages has been shown previously ; therefore , we believe that the ifn- originated from macrophages and that its production decreased after igf - i stimulation . although cytokine production was not altered by igf - i stimulation in amastigote - infected macrophages , when compared with the promastigote - infected macrophages , we observed differences in the production of il-1 and il-6 .
il-1 production was not altered in amastigote - infected macrophages but increased in promastigote - infected macrophages , and no changes were observed after igf - i stimulation .
we also observed increased il-6 upon igf - i stimulus in uninfected macrophages that remained increased at the same level in promastigote - infected cells but decreased in amastigote - infected cells to the level of the uninfected , unstimulated cells .
these data suggest that amastigotes suppress cytokine production in general . in line with our results ,
il-1 production was previously shown to be suppressed in leishmania donovani amastigote - infected human monocytes .
moreover , l. major amastigotes did not induce the production of the proinflammatory cytokine tnf and the chemokines ccl3 and ccl4 , which is in contrast to the higher production during promastigote infection .
similarly , with leishmania donovani amastigotes , a significant proportion of genes were shown to be suppressed in host cells .
no alteration in cytokine production that we observed in the amastigote - infected macrophages was also observed in a study with leishmania major where parasite arginase was further shown to affect host cell arginase activation .
this finding would be interesting to confirm with l. ( l. ) amazonensis but here we investigated ps exposure on tissue - derived l ( l. ) amazonensis amastigotes as it relates to the progression of the infection and is called apoptotic mimicry .
the induction factor was previously unknown , and because igf - i is present in tissue fluids and within macrophages which is the amastigotes niche , we considered the possible effect of this factor on ps exposure in amastigotes .
in fact , igf - i stimulation induced ps exposure on l. amazonensis amastigotes but , interestingly , not on promastigotes . a recent publication regarding leishmania promastigotes
shows the absence of ps in this form of the parasite although in our experiment with promastigotes the hydrogen peroxide induced exposure of ps or some similar molecule that has bound to anexin v ( data not shown ) .
however , we should emphasize that apoptotic mimicry and the present results refer to ps exposure on amastigotes , and the presence of ps is considered to be likely in other growth phases by the same authors . in the study on apoptotic mimicry ,
increased parasite growth was attributed to tgb- production ; however , in the present study , we explored the effect of ps exposure on arginase activity in macrophages . using ps - liposomes to restrict the analysis to this particular molecule
, we were able to show that in fact ps - liposomes but not the control pc - liposomes led to increased arginase activity in the macrophages .
furthermore , neutralization of tgf- using an anti - tgf- antibody had no effect on arginase activity .
of note , ps - liposomes were previously shown to have an effect on l - arginine metabolism , leading to a decrease in no production by lipopolysaccharide - stimulated macrophages . in the present study , we also observed decreased nitric oxide production but increased arginase activity after igf - i stimulation in amastigote - infected cells , suggesting the preferential activation of the alternative macrophage pathway , possibly due to ps exposure on amastigotes after stimulation .
further studies must be performed to clarify how ps - liposomes activate macrophage arginase , but because arginase is a trimeric metalloenzyme that contains a binuclear manganese cluster in the active site , we can speculate that the negative charge of the ps - liposome could carry manganese through cell membrane when ps - liposomes are internalized by macrophages , providing manganese to the enzyme and activating it .
studies using mouse and human cells show an increased leishmania parasite burden when apoptotic neutrophils are ingested by macrophages .
this increased burden occurs when using neutrophils derived from the leishmania major - susceptible balb / c mouse strain .
considering the data from the present study , we can speculate that this phenomenon may be related to the arginase activation induced by ps exposure on apoptotic cells . in the above - mentioned studies ,
the effect was related to the induction of pge2 and tgf- production [ 52 , 53 ] ; however , our data contradict this observation as a possible mechanism in the present experiments .
the present data suggest that in l. ( l. ) amazonensis amastigote - infected macrophages , igf - i induces arginase activity directly on amastigotes and also in macrophages .
the cytokine production data suggest that amastigote infection leads to the suppression of cytokine production with no contribution to macrophage activation upon igf - i stimulation .
a phenomenon that is seemingly peculiar to l. ( l. ) amazonensis is the induction of ps exposure on amastigotes , which was previously observed on tissue - derived amastigotes and was induced by igf - i and contributes to the progression of the infection in the present study .
finally , we should emphasize that the activation of macrophage arginase by ps - liposomes leads to the alternative activation of macrophages through cytokine - independent mechanisms . | leishmania ( leishmania ) amazonensis exhibits peculiarities in its interactions with hosts .
because amastigotes are the primary form associated with the progression of infection , we studied the effect of insulin - like growth factor ( igf)-i on interactions between l. ( l. ) amazonensis amastigotes and macrophages . upon stimulation of infected macrophages with igf - i , we observed decreased nitric oxide production but increased arginase expression and activity , which lead to increased parasitism .
however , stimulation of amastigote - infected macrophages with igf - i did not result in altered cytokine levels compared to unstimulated controls . because igf - i is present in tissue fluids and also within macrophages
, we examined the possible effect of this factor on phosphatidylserine ( ps ) exposure on amastigotes , seen previously in tissue - derived amastigotes leading to increased parasitism .
stimulation with igf - i induced ps exposure on amastigotes but not on promastigotes . using a ps - liposome instead of amastigotes
, we observed that the ps - liposome but not the control phosphatidylcholine - liposome led to increased arginase activity in macrophages , and this process was not blocked by anti - tgf- antibodies .
our results suggest that in l. ( l. ) amazonensis amastigote - infected macrophages , igf - i induces arginase activity directly in amastigotes and in macrophages through the induction of ps exposure on amastigotes in the latter , which could lead to the alternative activation of macrophages through cytokine - independent mechanisms . |
because mf is the disease of old age , coincidence with pregnancy is rare and only few cases have been reported .
until now there is controversy about the effect of pregnancy on progression of mf and some studies reported that pregnancy appeared to have no impact on the course of early mf , but according to our case report and some other reports , it seems that pregnancy may have some deleterious effect on the clinical course of mf .
mycosis fungoides ( mf ) , a subgroup of non - hodgkin 's t - cell lymphoma , is the most common type of primary cutaneous t - cell lymphoma , characterized by skin infiltration and occasionally systemic involvement .
in one retrograde study pregnancy appeared to have no impact on the course of early mf , and no adverse effect was noted on pregnancy .
this case report is about the deleterious effect of pregnancy on progression of mf , and until now this is the first case report about transformation of mf to cd30 + large t - cell lymphoma during pregnancy .
a 30 years old woman with history of scaling erythematous skin lesions since eight years ago was presented to mf clinic of alzahra hospital of isfahan university of medical sciences .
lesions first has been appeared on the scalp then spreaded to whole body ( erythroderma ) .
two years earlier skin biopsy has been done and histologic diagnosis of skin biopsy was dermatitis .
physical examination findings were : erythroderma with severe scaling , palmoplantar keratoderma imitating acquired ichthyosis and bilateral ectropion [ figure 1 ] .
two skin - biopsy was performed , the first specimen histologically was compatible with dermatitis , but the histologic evaluation of the second one confirmed the diagnosis of mf .
the ihc staining revealed that the infiltrating cells were cd2 + , cd4 + , cd8- , cd45ro+ , cd7- , cd30- .
exfoliative erythroderma with ectropion the criteria for diagnosis of mf was completed ( clinical criteria , ihc staining and tcr gene rearrangement analysis ) .
cbc , esr , crp , ldh , blood biochemistry , liver function tests and peripheral blood smear were within normal limits .
patient staged as stage iiia , at this stage our treatment plan was systemic puva therapy ( 3 session per week ) and interferon alfa 2b ( 3 million unit 3 session per weeks , sc ) two weeks later patient returned to our clinic and stated that she was pregnant . at this time
our treatment plan changed to nb - uvb therapy , but she rejected this plan and interrupted treatment until delivery .
ichthyosiform erythroderma with bilateral groin lymphadenopathy two weeks of antibiotic therapy was started but lymphadenopathy persisted .
[ figures 3 and 4 ] ihc staining of specimens revealed that infiltrating cells had the following markers cd2 + , cd5 + , cd4 + , cd8- , cd45ro+ , cd7- , alk1(- ) , cd30 strongly positive , ki67 positive in more than 90% [ figures 5 - 7 ] skin biopsy , epidermotropism of lymphocytes .
h and e , 100 the lymph node is almost completely effaced and replaced by large lymphocytes .
h and e , 400 lymph node cd30 , 100 bone marrow biopsy was normal .
previous skin specimens were re - evaluated and anaplastic large cell transformation of mf was confirmed .
incidence of mf is 0.3 per 100,000 person per year and , it is twice more common in men than women .
the mean age of diagnosis in western countries is approximately 60 - 80 years . in this region of the world
( iran , isfahan ) the median age of mf patients is 43.14 years and the male : female ratio is 3:4 ( in contrast to reports from other countries ) .
these epidemiologic characteristics would partially explain the occasional coincidence of pregnancy and mf in our patients in isfahan iran . in 1981 , vonderheid , et al . reported a woman with a long - standing eczematous eruption that her skin lesions exacerbated during each of her three uncomplicated pregnancies .
three years after her last pregnancy , the eruption was diagnosed as mf developing into the tumour stage . in 2001 castelo - branco ,
et al . described a women suffering from stage iv - b mf who became pregnant three years after the disease had been diagnosed . in spite of interruption of her therapy during pregnancy ,
the pregnancy had no adverse effect on the course of mf . in another case report in 2001
echols , et al . described a 26-year - old black woman diagnosed to be suffering from mf ( the stage was not noted ) 1 year before she became pregnant . at the beginning of her third trimester , she presented with an acute exacerbation of mf , so she was treated with puva and topical steroids , with complete remission . on the other hand , in the report of iris amitay , et al .
, in a series of seven women with early - stage mf , there was no evidence indicating that pregnancy changed the course of the disease . in two mf patients that each undergone two pregnancies during the course of their disease , this finding was consistent and pregnancy appeared to have no impact on the course of early mf .
of ten cases of mf during pregnancy described above , two showed progression during gestation , but none of them had large cell transformation .
patients with plaque - type or erythrodermic mf may develop cutaneous tumors with large cell histology .
the cells of this tumor are at least four times greater in size than a small lymphocyte .
when over 25% of mf cells changes to large cell or when small nodules of these cells are identified on histology the transformation is diagnosed .
incidence rates of transformation in patients with mf have ranged from approximately 10 to 25% ; with variation according to the stage of mf at presentation . in one study ,
the cumulative probability of transformation in stages ii - b and iv of disease was estimated to be about 33% . in this study elevation of serum beta-2 microglobulin and/or lactate dehydrogenase ( ldh ) ,
mentioned as predictive for transformation ( p = 0.009 ) . in another study of 419 patients with mf ,
45 were found to have transformed at a median time after diagnosis of 6.5 years .
our case staged as iii - a , with normal ldh at presentation which during pregnancy progressed to anaplastic large cell lymphoma . on the basis of reports mentioned above
, it seems that this transformation was too rapid and it seems that pregnancy had a deleterious effect on the course of mf in this patient .
this case report suggests a probable relation between cd30 + large cell transformation of mf and pregnancy .
it is better to discuss about the probable risk of pregnancy in young women with mf who are planning for pregnancy .
this is the first case report of cd30 positive large cell transformation of mf during pregnancy . | mycosis fungoides ( mf ) a cutaneous t - cell lymphoma , is a subgroup of non - hodgkin 's lymphomas , characterized by skin infiltration and occasionally systemic involvement .
mf coincidence with pregnancy is rare .
the effect of pregnancy on mf and the effect of this disease on pregnancy are still unknown .
there are few case reports about pregnancy and its deleterious effect on the clinical course of mf .
this case report is about a 30-years - old female with mf who became pregnant and after delivery developed cd30 + large cell transformation ; this is the first report of large cell transformation of mf related to pregnancy . |
we reviewed leptospirosis case investigation reports by hawaii department of health ( hdoh ) investigators submitted during 19992008 .
these reports were ( and still are ) generated for all reported leptospirosis cases in the state .
a standardized case investigation form was used , which includes demographic , epidemiologic , clinical , and laboratory information obtained from patient interviews , medical record reviews , and laboratory reports .
research for this study was approved by the hdoh institutional review board . for exposure source to be assessed , incubation periods estimated , and exposures classified , patients were asked about high - risk activities that occurred during the 21 days before symptom onset .
these included exposure to animals , mud , or potentially contaminated freshwater sources involving occupational activities ( e.g. , farming , ranching ) , recreational activities ( e.g. , freshwater swimming , hiking ) , or habitational activities ( around the home ; e.g. , gardening , trapping rats ) . if exposure was continuous or if persons had been exposed multiple times , the incubation period was considered indeterminate .
ascertainment of exposure classification involved placing cases into 3 mutually exclusive exposure categories : occupational , recreational , or habitational . if exposure activities involved > 1 category , the exposure classification was considered indeterminate .
outbreaks were defined as > 2 epidemiologically linked cases . a patient with a confirmed case had a clinically compatible illness plus a > 4-fold increase in microscopic agglutination test ( mat ) titer between acute- and convalescent - phase serum specimens or isolation of leptospira spp . from a clinical specimen ( 6,7 ) .
all other cases were classified as either probable ( clinically compatible illness with mat titer > 200 in > 1 serum specimens without a 4-fold increase in titer ) or suspected ( clinically compatible illness with less supportive laboratory evidence of infection [ e.g. , mat titer < 200 , positive macroscopic slide agglutination test result , reactive immunoglobulin ( ig ) m elisa , or positive indirect hemagglutination assay results ] ) . only laboratory - confirmed cases in patients whose disease was contracted through exposure within the state of hawaii were included in this analysis .
mats were conducted by cdc from january 1999 through november 2004 , and by hdoh from december 2004 through december 2008 .
all isolates were sent to cdc for definitive serogroup identification . to determine the presumptive infecting serogroup for serologically confirmed cases ,
if > 1 serogroup had the same high titer , the identification was labeled indeterminate . to calculate mean annual incidence rates ( overall and by age , sex , and hawaii island on which patient was exposed ) , the numerator was the number of cases for the specified groups over the 10-year observation period divided by 10 .
the denominator was the overall or relevant group - specific population estimate from the 2000 us census ( 9 ) .
data from our earlier 25-year study period , 19741998 , were used for trend analyses ( 5 ) .
we calculated frequencies , tests for trends , and tests for difference using epi info version 3.3.2 ( cdc , atlanta , ga , usa ) ; p values < 0.05 were considered significant .
hdoh received 356 leptospirosis case reports ; 345 were related to exposures within the state of hawaii .
the 11 cases from exposures occurring out of state included 2 from guam ; 2 from thailand ; and 1 each from panama , the federated states of micronesia , borneo , okinawa , malaysia , singapore , and texas . of the case
reports related to in - state exposures , 198 ( 57% ) were laboratory confirmed , 116 ( 34% ) were probable , and 31 ( 9% ) were suspected .
the number of confirmed cases reported per year ranged from 11 to 27 ( median 20 ) , and the estimated mean annual incidence rate was 1.63 per 100,000 population . mean monthly reported cases were highest from october through february ( figure 1 ) .
the observed seasonal disease occurrence for the recent 10-year study period was significantly different from that of the previously reported 25-year study period ; summer cases predominated in the latter ( p<0.01 ) ( 5 ) .
month of onset for 198 laboratory - confirmed leptospirosis cases , hawaii , usa , 19992008 .
case - patients were predominately male ( 91% ) , and ages ranged from 3 to 76 years ( median 38 years ) .
the highest age - specific rate was among persons 2029 years of age , and the lowest was among children 09 years of age .
most cases and the highest incidence rates were related to exposures on the islands of kauai and hawaii ( table 1 ) .
in addition , cases were most consistently reported from the northeast , windward sides of the islands : hanalei ( n = 8) and wailua ( n = 12 ) on kauai , waipio valley ( n = 12 ) and hilo ( n = 17 ) on hawaii , and maunawili falls ( n = 13 ) on oahu ( figure 2 ) .
case - patients observed over 10 years for the specified category divided by 10 divided by specified subgroup population estimate from 2000 us census data ( 9 ) .
exposure locations associated with the greatest number of leptospirosis cases , hawaii , usa , 19992008 .
we were able to determine exposure classifications for 177 ( 89% ) of the 198 confirmed cases .
recreational exposures accounted for 79 ( 45% ) and were mostly related to freshwater swimming , hiking , and camping .
occupational exposures accounted for 78 ( 44% ) , mostly relating to farming , specifically , taro farming .
exposures around the home accounted for 20 ( 11% ) , most commonly , gardening . after categorizing cases into 5-year intervals and comparing the results with reports from 1989 through 1998 ( 5 )
, we found that recreational exposures remained relatively stable over the past 20 years ( 19892008 ) , while occupational exposures actually increased , but the difference was not significant ( p = 0.08 ) ( figure 3 ) .
after stratification by island , a significant increase in occupational exposures was shown for the island of hawaii ( p = 0.04 ) .
trends in exposure classification for laboratory - confirmed leptospirosis cases , hawaii , usa , 19892008 . most cases occurred sporadically .
one outbreak ( > 2 epidemiologically linked cases ) , which involved 2 landscapers , occurred on kauai in 1999 ; both cases were laboratory confirmed .
another outbreak ( 2 epidemiologically linked cases : 1 confirmed , 1 probable ) was associated with flooding of the university of hawaii campus on october 31 , 2004 , when heavy rains caused an adjacent stream to overflow its banks ( 10 ) . for case - patients with known exposure dates , the median incubation period was 9 days ( range 121 days ) .
a total of 118 ( 73% ) of 161 case - patients , for whom treatment information was available , were hospitalized .
the most frequent signs and symptoms among patients who sought treatment were fever , myalgias , headache , nausea , and vomiting .
abnormal urinalysis results were common ; specimens from 78 ( 73% ) of 107 and 71 ( 68% ) of 105 case - patients showed hematuria and proteinuria , respectively .
results of liver function tests were frequently abnormal as well ; laboratory results for 109 ( 74% ) of 147 case - patients showed elevated alanine aminotransferase levels ( > 40
u / l ) , and 85 ( 63% ) of 134 showed elevated total bilirubin levels ( > 1 mg / dl ) .
the most common hematologic anomaly was thrombocytopenia ( < 140 10/l ) , which was observed for 97 ( 66% ) of 146 case - patients ( table 2 ) .
the most common initial diagnosis was leptospirosis for 114 ( 75% ) of 151 patients . during the 10-year reporting period ,
1 death occurred among 198 patients with confirmed cases ( case - fatality rate 0.5% ) .
a 23-year - old man who attended college on the mainland had been exposed through recreational activities while at home in hawaii during winter break 2003 .
of the 198 patients with confirmed infection , 152 ( 77% ) received a diagnosis on the basis of serologic testing with the mat , 18 ( 9% ) cases were confirmed with culture isolates , and 28 ( 14% ) were confirmed by mat and isolates .
forty - three isolates obtained during 20002008 were characterized at cdc by molecular and serologic techniques .
isolates were grouped into 4 clades based on mat results and pulsed - field gel electrophoresis : 19 ( 44% ) unknown serovar ( serogroup australis ) , 17 ( 40% ) serovar icterohemorrhagiae ( serogroup icterohemorrhagiae ) , 4 ( 9% ) serovar ballum ( serogroup ballum ) , and 3 ( 7% ) of unknown serovar ( serogroup bataviae ) .
cross - agglutination absorption assay identified the unknown serovar from serogroup australis as a new serovar closely related to lora ( 11 ) .
the most common infecting serogroups ( identified definitively by isolate or presumptively by mat ) were australis ( n = 50 ) and icterohemorrhagiae ( n = 51 ) .
analysis for linear trend , after cases were categorized into 5-year intervals and compared with confirmed cases reported during 19741998 ( 5 ) , showed a significant increase in infections attributed to serogroup australis and a decrease in infections caused by serogroup icterohemorrhagiae ( p<0.0001 for each ) .
the most recent 10-year reporting period has demonstrated a statistically significant shift in the seasonal occurrence of leptospirosis from the drier summer months ( 5 ) to the wetter winter months .
climatologists have characterized the hawaiian archipelago as having only 2 seasons : summer ( may through september ) and winter ( october through april ) .
rainfall and widespread rainstorms are most common during the winter months ( 12 ) . during the earlier reporting period , 19741998 ,
recreationally associated exposures predominated and increased over time ; therefore , the summer predominance was attributed to the greater likelihood of recreational exposure in the summer . during 19892008 , the frequency of recreational exposures plateaued while frequency of occupational exposures seemed to increase .
this observed change in exposure history might allow seasonal climatic effect to have a greater influence on the epidemiology of the disease .
in addition , taro farming , a recognized high - risk occupation ( 5 ) , which had been on the decline , has experienced a resurgence relating to renewed interest in the cultural importance to native hawaiians and an awareness of taro s nutritional value ( 13 ) . in 2000 , hawaii produced 7 million pounds of taro , the largest crop yield since 1977 ( 14 ) .
the island distribution of leptospirosis cases remains virtually unchanged since our earlier report ( 5 ) .
kauai , the island with the highest annual rainfall and second most rural island , had and continues to have the highest incidence rate , followed by hawaii , the most rural island .
as in our earlier report , cases were most consistently reported from the wetter , windward , northeast sides of each island .
notably , climatic changes have been documented for the hawaiian archipelago with significant trends in increasing temperatures ( 15 ) , decreasing rainfall ( 16 ) , and increasing rain intensity ( 17 ) over the past 30 years .
the effects of climate change on ecosystems are complex , but the potential for influencing infectious disease patterns has been well described ( 18,19 ) .
temperature and climate changes may affect the host animal s environment , making transmission to humans more likely .
increase in rain intensity with resultant flooding is a well - recognized climatic risk factor for transmission of leptospira spp ( 20 ) .
flooding was responsible for 1 of the 2 outbreaks during the study period ( 10 ) .
the predominance of men among case - patients is well recognized ( 2124 ) and is virtually unchanged from our earlier report ( 5 ) .
this predominance has been explained by the tendency of more men to participate in high - risk outdoor exposure activities .
the low reported age - specific case rates in children < 10 years of age and highest rates among adults 2050 years of age are also consistently reported ( 2224 ) and similar to our earlier findings ( 5 ) .
our findings corroborate other large case series that show that the most common clinical manifestation of leptosporisis are nonspecific signs or symptoms , such as fever , headache , and mylagias ( 5,22,2527 ) . the case - fatality rate ( 0.5% ) is lower than that reported from brazil ( 25 ) , barbados ( 28 ) , guadeloupe ( 22 ) , and the andaman islands ( 26 ) , but similar to the rates found in our earlier study ( 5 ) and in a recent case series from france ( 27 ) .
the low case - fatality rate in this series may be explained by early recognition and initiation of supportive therapy and antimicrobial drugs . other case series may be biased toward recognition and inclusion of only the most severely ill , hospitalized patients , which leads to higher case - fatality rates .
a recent population - based case - control study from brazil ( 29 ) showed that pulmonary involvement was the strongest independent predictive factor for death caused by severe leptospirosis .
pulmonary findings were infrequent among case - patients in this study , the earlier hawaii series ( 5 ) , and the france series ( 27 ) .
the changing temporal trend in the infecting serogroup first identified in our earlier study has continued ; most of the current leptospirosis isolates are in the australis serogroup . this documented trend over the past 35 years from the previously predominant serogroup icterohemorrhagiae to the now predominant australis may reflect the influence of different host animals , the effects of climatic and land use changes , or both .
serogroup icterohemorrhagiae has been associated with rats ( rattus norwegicus and r. rattus ) , and australis has been associated with swine , including feral swine or wild boars ( sus scrofa ) ( 30,31 ) .
recent reports from germany have shown high seroprevalence of australis serogroup ( serovar bratislava ) in urban feral swine ( 32 ) and documented increased size in the feral swine population and habitat changes leading to epidemiologic linkages between leptospirosis occurrence and feral swine exposure ( 33 ) .
hawaii has also experienced an increase in the feral swine population , with a concordant sharp increase in the number of feral swine encroaching on urban residential areas ( 34,35 ) .
researchers at the university of hawaii are currently investigating the possible influence of feral swine exposure on human disease in hawaii by undertaking a leptospirosis seroprevalence study of feral swine .
annual reported leptospirosis incidence rates in the united states ranged from 0.02 to 0.05 per 100,000 population from 1974 through 1994 , the last year leptospirosis was included in the list of nationally notifiable diseases ( 5 ) .
if we include probable and suspected cases , as was done nationally , our mean estimated annual incidence rate during this 10-year study period would increase from 1.63 to 2.85 per 100,000 population , 100 greater than that reported nationally .
compared with other locales for which annual leptospirosis incidence rates are available , hawaii would be considered in the moderate range category ( 110/100,000 population ) ( 36 ) .
countries in this range include cuba ( 2.47/100,000 population ) and costa rica ( 6.72/100,000 population ) ( 3 ) .
countries categorized as having high rates ( > 10/100,000 population ) include barbados ( 10.03/100,000 population ) , trinidad and tobago ( 12.04/100,000 population ) , and seychelles ( 43.21/100,000 population ) ( 3 ) .
additional countries or regions considered to have high rates ( for which data are not available ) are vietnam and french polynesia ( 36 ) . although leptospirosis is a notifiable disease in hawaii , case reporting is based on passive surveillance and likely underestimates true disease occurrence . during a 1-year period in 1988 and 1989 ,
an active surveillance study was conducted on the islands of hawaii and kauai , which resulted in a 5-fold increase in case identification ( 37 ) .
a recent retrospective analysis of serum obtained from febrile patients during a dengue fever outbreak in hawaii , 20012002 , also identified a substantial number of leptospirosis cases that otherwise would have gone undiagnosed ( 38 ) .
future field studies using geographic information system technology to link climatic and environmental phenomena , such as rainfall occurrence and environmental isolates with human and animal infection , could offer valuable insights .
given the potential effects of climate and land use changes , public health officials must remain alert to the occurrence and changing epidemiology of emerging and reemerging infectious diseases . without national surveillance , the occurrence of leptospirosis outside of hawaii or other regions that have leptospirosis surveillance may go largely unrecognized , and thus , unmonitored .
ongoing surveillance activities , such as ecologic , animal , and laboratory studies are necessary to clarify and track the dynamic epidemiology of this widespread , reemerging zoonotic illness . | although infrequently diagnosed in the united states , leptospirosis is a notable reemerging infectious disease throughout developing countries . until 1995 ,
when the disease was eliminated from the us list of nationally notifiable diseases , hawaii led the nation in reported annual incidence rates .
leptospirosis remains a notifiable disease in hawaii . to ascertain the status of leptospirosis in hawaii since the most recent us report in 2002 , we reviewed 19992008 data obtained from case investigation reports by the hawaii state department of health .
of the 345 case reports related to in - state exposures , 198 ( 57% ) were laboratory confirmed .
our findings indicate a change in seasonal disease occurrence from summer to winter and in the infective serogroup from icterohemorrhagiae to australis .
also , during the past 20 years , recreational exposures have plateaued , while occupational exposures have increased .
ongoing surveillance is needed to clarify and track the dynamic epidemiology of this widespread zoonosis . |
corneal transplantation for corneal endothelial diseases is undergoing a paradigm shift from penetrating keratoplasty to endothelial keratoplasty.1 descemet s stripping automated endothelial keratoplasty ( dsaek ) , which is essentially endothelial keratoplasty that involves donor tissue preparation using an automated microkeratome , is rapidly becoming the preferred alternative to conventional penetrating keratoplasty for endothelial dysfunction , such as fuchs endothelial keratoplasty and pseudophakic bullous keratopathy.2 in the united states , the number of donor corneas for endothelial keratoplasty has exponentially increased from 3% ( 2005 ) to 33% ( 2007 ) and 42.8% in 2009 ( eye banking statistical report 2009 , eye bank association of america data ( http://www.restoresight.org/donation/statistics.htm ) .
the advantages of dsaek over penetrating keratoplasty include better tectonic stability , essentially sutureless surgery , and faster postoperative visual rehabilitation with more predictable refractive changes.37 however , early studies mainly in caucasian eyes suggest equivalent or higher cell loss in dsaek compared to penetrating keratoplasty at 6 months , albeit nonsignificant by 2 years.3,812 the main aim of this study was to compare our dsaek results in terms of endothelial cell loss with those of penetrating keratoplasty with at least 1-year follow - up in asian eyes using our previously described dsaek technique.13
we conducted a retrospective study of patients who underwent dsaek or penetrating keratoplasty for which the surgical indication was either fuchs endothelial dystrophy or pseudophakic and aphakic bullous keratopathy in 20062008 .
we excluded any patients who did not have a minimum pos - surgical follow - up of 1 year .
our subjects and clinical data were obtained from the ongoing cohort of the singapore corneal transplant study , an audited longitudinal prospective study which contains preoperative , intraoperative , and yearly postoperative follow - up clinical data.14 this study followed the principles of the declaration of helsinki , with ethics approval obtained from our institutional review board .
a total of 241 patients met our inclusion criteria , of whom 68 underwent dsaek and 173 underwent penetrating keratoplasty by the five corneal surgeons at our center , as well as inclusion of cases which were partially performed by corneal fellows in training under direct supervision .
our main outcome measure was endothelial cell count and the derived percent endothelial cell loss at 1 year follow - up .
our secondary outcome measures included graft success and visual acuity at 1 year follow - up .
visual acuity was measured using the snellen visual acuity chart and we analyzed the results using logarithm of the minimum angle of resolution ( logmar ) equivalent units , including manifest refraction , spherical equivalent , and cylindrical error.15 the singapore eye bank provides all donor corneas , stored in optisol in cold storage , with standard internal guidelines for penetrating keratoplasty and dsaek grafts and we obtained all donor information from their database , including donor endothelial cell counts.14 preoperative specular microscopy of the donor tissue was performed either by certified technicians in an eye bank association of america- certified eye bank or by a certified eye bank technician at the singapore eye bank .
postoperative specular microscopy measurements of endothelial cell density were performed using a noncontact specular microscope ( konan medical corporation , hyogo , japan ) at 12 months postoperatively , by ophthalmic technicians trained in specular microscopy .
calibrations and magnifications were standardized automated measurements with a mean value derived , as previously described.16 the incidence of postoperative complications was obtained from the singapore corneal transplant study database , which tracks all graft complications .
graft failure was defined as irreversible loss of optical clarity , with the date of onset of corneal clouding selected as the time point of graft failure .
penetrating keratoplasty surgeries were performed using a standard technique based on a hanna vacuum trephine system ( moria inc , antony , france ) . in summary , the recipient cornea was excised using the hanna trephine .
a 0.250.50 mm oversized donor cornea was then punched out endothelial side up and sutured on to the recipient with 10 - 0 nylon , using either an 8-bite , 10 - 0 nylon double continuous running suture or a combination of a single 8-bite 10 - 0 nylon continuous and eight interrupted sutures .
a bandage contact lens was placed at the end of the surgery , and subconjunctival dexamethasone 0.1% ( decadron ; merck and co , inc , rahway , nj ) , gentamicin 14 mg / ml ( garamycin ; schering ag , berlin - wedding , germany ) , and cefazolin 50 mg / ml ( ancef ; glaxosmithkline , research triangle park , nc ) was injected .
dsaek was performed using our previously described technique.13,16 essentially , after descemet s stripping under air,17 a paracentesis was first made in the peripheral cornea opposite the scleral tunnel wound for insertion of kawai intraocular capsulorhexis forceps ( asico , westmont , il ) or tan dsaek forceps ( asico).13 a standard anterior chamber intraocular lens sheets glide ( bd visitec ) was trimmed to 4.5 mm and inserted into the eye through a 5 mm temporal scleral tunnel incision while the anterior chamber was maintained via an anterior chamber maintainer with a balanced salt solution infusion .
the donor was prepared by the surgeon using an automated lamellar therapeutic keratoplasty system ( altk ; moria sa , antony , france ) aiming for a donor cornea of thickness of approximately 150 microns .
four preplaced corneal venting incisions were made on the recipient cornea . a dispersive ophthalmic viscosurgical device ( viscoat ; alcon laboratories inc , hnenberg , switzerland ) was liberally applied over the endothelial surface of the donor cornea and on the anterior surface of the glide , taking care not to have the ophthalmic viscosurgical device on the stromal donor surface .
the donor cornea was gently inverted , corneal endothelial surface - down , onto the ophthalmic viscosurgical device covered portion of the glide .
kawai or tan dsaek forceps were passed through the nasal paracentesis , over the sheets glide , and out through the scleral incision , grasping the donor cornea stromal edge and pulling the donor cornea through the scleral incision , whilst the anterior chamber maintainer was infusing balanced salt solution at a medium to slow rate . with this technique ,
a small air bubble was injected under the donor cornea with a 30-gauge canula to prevent descent of the donor cornea , the sheets glide retracted , and the donor cornea was released from the forceps .
the scleral tunnel was then sutured with three 10/0 nylon interrupted sutures , the anterior chamber maintainer was removed and the port sutured , the donor was adjusted centrally by gentle massage through the cornea surface , and full air tamponade was achieved with a large bubble in the anterior chamber for 8 minutes . following this , some air was replaced with balanced salt solution , leaving a smaller air bubble approximating the size of the endothelial keratoplasty graft in the anterior chamber .
all patients were examined approximately one hour after surgery to ensure air was still present in the anterior chamber , and no donor dislocation or pupillary block was present .
we used similar postoperative medication regimens in both groups , ie , predforte ( prednisolone acetate ophthalmic suspension , usp ) 1% every 3 hours for 1 week , three times a day for 6 months , twice daily for 3 months then once a day for up to 1 year . statistical analysis
included descriptive statistics , whereby the mean and standard deviation were calculated for the continuous variables , while the frequency distribution and percentages were used for categorical variables .
comparisons between categorical variables were conducted using fisher s exact tests , whereas the one - way analysis of variance test was used for means .
meier survival analysis was conducted to determine survival probabilities of penetrating keratoplasty and dsaek groups .
the survival period of failed grafts was defined as the time between the date of surgery and recorded date of survival or failure .
penetrating keratoplasty surgeries were performed using a standard technique based on a hanna vacuum trephine system ( moria inc , antony , france ) . in summary , the recipient cornea was excised using the hanna trephine .
a 0.250.50 mm oversized donor cornea was then punched out endothelial side up and sutured on to the recipient with 10 - 0 nylon , using either an 8-bite , 10 - 0 nylon double continuous running suture or a combination of a single 8-bite 10 - 0 nylon continuous and eight interrupted sutures .
a bandage contact lens was placed at the end of the surgery , and subconjunctival dexamethasone 0.1% ( decadron ; merck and co , inc , rahway , nj ) , gentamicin 14 mg / ml ( garamycin ; schering ag , berlin - wedding , germany ) , and cefazolin 50 mg / ml ( ancef ; glaxosmithkline , research triangle park , nc ) was injected .
dsaek was performed using our previously described technique.13,16 essentially , after descemet s stripping under air,17 a paracentesis was first made in the peripheral cornea opposite the scleral tunnel wound for insertion of kawai intraocular capsulorhexis forceps ( asico , westmont , il ) or tan dsaek forceps ( asico).13 a standard anterior chamber intraocular lens sheets glide ( bd visitec ) was trimmed to 4.5 mm and inserted into the eye through a 5 mm temporal scleral tunnel incision while the anterior chamber was maintained via an anterior chamber maintainer with a balanced salt solution infusion .
the donor was prepared by the surgeon using an automated lamellar therapeutic keratoplasty system ( altk ; moria sa , antony , france ) aiming for a donor cornea of thickness of approximately 150 microns .
four preplaced corneal venting incisions were made on the recipient cornea . a dispersive ophthalmic viscosurgical device ( viscoat ; alcon laboratories inc , hnenberg , switzerland ) was liberally applied over the endothelial surface of the donor cornea and on the anterior surface of the glide , taking care not to have the ophthalmic viscosurgical device on the stromal donor surface .
the donor cornea was gently inverted , corneal endothelial surface - down , onto the ophthalmic viscosurgical device covered portion of the glide .
kawai or tan dsaek forceps were passed through the nasal paracentesis , over the sheets glide , and out through the scleral incision , grasping the donor cornea stromal edge and pulling the donor cornea through the scleral incision , whilst the anterior chamber maintainer was infusing balanced salt solution at a medium to slow rate . with this technique ,
a small air bubble was injected under the donor cornea with a 30-gauge canula to prevent descent of the donor cornea , the sheets glide retracted , and the donor cornea was released from the forceps .
the scleral tunnel was then sutured with three 10/0 nylon interrupted sutures , the anterior chamber maintainer was removed and the port sutured , the donor was adjusted centrally by gentle massage through the cornea surface , and full air tamponade was achieved with a large bubble in the anterior chamber for 8 minutes . following this ,
some air was replaced with balanced salt solution , leaving a smaller air bubble approximating the size of the endothelial keratoplasty graft in the anterior chamber .
all patients were examined approximately one hour after surgery to ensure air was still present in the anterior chamber , and no donor dislocation or pupillary block was present .
we used similar postoperative medication regimens in both groups , ie , predforte ( prednisolone acetate ophthalmic suspension , usp ) 1% every 3 hours for 1 week , three times a day for 6 months , twice daily for 3 months then once a day for up to 1 year .
statistical analysis included descriptive statistics , whereby the mean and standard deviation were calculated for the continuous variables , while the frequency distribution and percentages were used for categorical variables .
comparisons between categorical variables were conducted using fisher s exact tests , whereas the one - way analysis of variance test was used for means .
meier survival analysis was conducted to determine survival probabilities of penetrating keratoplasty and dsaek groups .
the survival period of failed grafts was defined as the time between the date of surgery and recorded date of survival or failure .
overall , there were more patients whose main surgical indication was bullous keratopathy as compared with fuchs dystrophy ( 153/241 , 63.5% ) and more of the patients with bullous keratopathy had undergone a penetrating keratoplasty as compared with dsaek ( 120/173 , 69.4% versus 33/68 , 48.5% ; p = 0.003 ) .
however , there were no significant differences in demographics or characteristics of patients with bullous keratopathy or fuchs dystrophy between the treatment groups .
the mean preoperative donor endothelial cell density was 101 cells / mm greater in the dsaek than in the penetrating keratoplasty group ( 2792 327 versus 2691 360 cells / mm ; p = 0.0412 ) .
donor size was significantly larger in patients undergoing dsaek as compared with penetrating keratoplasty ( mean donor size 8.75 0.49 versus 7.87 0.34 mm ; p < 0.001 ) .
postoperative endothelial cell density was obtainable in 120 eyes ( 60 dsaek - treated eyes and 60 penetrating keratoplasty - treated eyes ) performed at 1-year follow - up . in this subanalysis
, there were no significant differences in baseline characteristics , such as mean recipient age ( 64 10 versus 67 11 years ; p = 0.08 ) sex ( p = 0.06 ) , race ( p = 0.16 ) , or diagnosis ( 51% versus 49% fuchs dystrophy ; p = 0.86 ) between the dsaek and penetrating keratoplasty groups .
we found that mean endothelial cell density was greater at 1 year in the dsaek group as compared with the penetrating keratoplasty group ( 2174 66 versus 1555 76 ; p = 0.001 ) and overall percentage of endothelial cell loss after 1 year was 40.9% 2.9% in penetrating keratoplasty - treated and 22.4% 2.3% in dsaek - treated eyes ( p < 0.001 ) . in patients with fuchs dystrophy
, dsaek - treated eyes had significantly less percentage of endothelial cell loss compared with penetrating keratoplasty - treated eyes ( 30 eyes each group , mean percentage endothelial cell loss ; 20.4% 1.4% versus 37.7% 2.3% ; p = 0.001 ) .
this was similar to patients with bullous keratopathy ( 30 eyes each group , mean percentage of endothelial cell loss ; 24.2% 2.0% versus 41.8% 2.2% ; p = 0.002 ) .
we also compared percentage of endothelial cell loss at 1 year between patients with fuchs dystrophy and bullous keratopathy . at 1 year , the percentage of endothelial cell loss did not differ significantly between patients with fuchs dystrophy and those with bullous keratopathy for either procedure ( 39% versus 44% in penetrating keratoplasty - treated eyes ; p = 0.40 ; and 20% versus 24% in dsaek - treated eyes ; p = 0.45 ) . in our study cohort ,
26 patients had concomitant eye disease , which significantly impacted on final visual outcome , while 17 patients had late graft failure at 1 year .
all these patients were excluded from our final visual acuity analysis ( table 2 ) .
we compared the remaining patients ( 61 dsaek with 137 penetrating keratoplasty - treated eyes ) with respect to visual acuity . amongst these patients
there were fewer patients who had fuchs dystrophy in the penetrating keratoplasty group ( 35/61 [ 57.3% ] dsaek ; 51/137 [ 37.2% ] penetrating keratoplasty ; p = 0.011 ) .
patients who underwent dsaek had better visual outcomes at 1 year when compared with penetrating keratoplasty - treated patients ( table 3 ) .
overall , dsaek - treated eyes had significantly superior uncorrected visual acuity ( ucva ) and best spectacle - corrected visual acuity ( bscva ) as compared with penetrating keratoplasty - treated eyes at 1 year ( mean difference ucva , 0.42 0.0059 ; p < 0.001 ; bscva , 0.14 0.032 ; p < 0.001 ) .
in addition , there were more eyes with bscva better than 20/40 in dsaek - treated group as compared with the penetrating keratoplasty - treated group ( 67.2% versus 38.7% ; p < 0.001 ) .
when comparing visual outcomes between surgical indications , we found that visual outcome was significantly better in eyes with fuchs dystrophy for both dsaek ( mean difference bscva , 0.36 0.011 ; p = 0.002 ) and penetrating keratoplasty ( mean difference bscva , 0.40 0.0079 ; p < 0.001 ) .
overall , patients with penetrating keratoplasty - treated eyes had higher astigmatism at 1 year as compared with dsaek - treated eyes ( 3.0 2.1 versus 1.7 0.8 , respectively ; p < 0.001 , table 1 ) .
we also observed this difference in patients with fuchs dystrophy ( 2.6 1.8 versus 1.7 0.8 ; p = 0.023 ) and bullous keratopathy ( 3.2 2.2 versus 1.7 0.8 ; p = 0.001 , table 3 ) .
overall , dsaek - treated eyes were more hyperopic as compared with penetrating keratoplasty - treated eyes at 1 year ( spherical equivalent + 1.5 3.3 versus 0.25 1.8 , respectively ; p < 0.001 ) .
we had one case of primary graft failure in each of the dsaek and penetrating keratoplasty treatment groups ( 1/68 , 1.5% versus 1/173 , 0.5% respectively ; p = 0.31 ) . at 1 year , 66/68 ( 97.0% ) eyes that underwent dsaek had clear grafts while 158/173 ( 92.0% ) of penetrating keratoplasty - treated eyes had clear grafts ( p = 0.479 ) .
there were no significant differences in late graft failure between the groups ( penetrating keratoplasty 8% versus dsaek 3% ; p = 0.118 ) .
reasons for graft failure in the penetrating keratoplasty group were infection - related ( n = 10 ) and immune - related ( n = 5 ) ; and for the dsaek group were immune - related ( n = 2 ) .
the kaplan meier probability of survival at 1 year was 95.3% for the penetrating keratoplasty - treated group , which decreased to 89.6% at 18 months , while it was 98.4% at 1 year and 93.2% for dsaek cases up to 18 months of follow - up ( p < 0.001 ) .
complications during the 1-year follow - up period were recorded according to our singapore corneal transplant study guidelines.14 we scored complications as events and made comparisons between the penetrating keratoplasty and dsaek groups ( table 4).18 epitheliopathy was significantly higher in penetrating keratoplasty - treated eyes ( p = 0.014 ) , while transient episodes of intraocular pressure elevation > 21 mmhg ( defined in terms of short - term , ie , 3 months use of antiglaucoma medications ) were seen in 20/68 ( 29.4% ) and 52/173 ( 30.0% ) for dsaek and penetrating keratoplasty , respectively ( p = 0.93 ) . of note , most of these patients ( 16/20 [ 80.0% ] for dsaek and 49/52 [ 94.2% ] for penetrating keratoplasty ) had an underlying history of glaucoma ( p = 0.07 )
. only four eyes ( three penetrating keratoplasty , one dsaek ) had had trabeculectomy performed previously , and there were no significant differences in graft outcomes .
all eyes with transiently raised intraocular pressure were treated successfully with intraocular pressure - lowering topical and/or systemic medications .
one patient who underwent dsaek had an acute graft rejection episode successfully treated with topical steroids , as compared with 11 ( 6.3% ) in the penetrating keratoplasty - treated group .
of note , we had no graft dislocations in our dsaek - treated eyes in this series of patients .
overall , there were more patients whose main surgical indication was bullous keratopathy as compared with fuchs dystrophy ( 153/241 , 63.5% ) and more of the patients with bullous keratopathy had undergone a penetrating keratoplasty as compared with dsaek ( 120/173 , 69.4% versus 33/68 , 48.5% ; p = 0.003 ) .
however , there were no significant differences in demographics or characteristics of patients with bullous keratopathy or fuchs dystrophy between the treatment groups .
the mean preoperative donor endothelial cell density was 101 cells / mm greater in the dsaek than in the penetrating keratoplasty group ( 2792 327 versus 2691 360 cells / mm ; p = 0.0412 ) .
donor size was significantly larger in patients undergoing dsaek as compared with penetrating keratoplasty ( mean donor size 8.75 0.49 versus 7.87 0.34 mm ; p < 0.001 ) .
postoperative endothelial cell density was obtainable in 120 eyes ( 60 dsaek - treated eyes and 60 penetrating keratoplasty - treated eyes ) performed at 1-year follow - up . in this subanalysis , there were no significant differences in baseline characteristics , such as mean recipient age ( 64 10 versus 67 11 years ; p = 0.08 ) sex ( p = 0.06 ) , race ( p = 0.16 ) , or diagnosis ( 51% versus 49% fuchs dystrophy ; p = 0.86 ) between the dsaek and penetrating keratoplasty groups .
we found that mean endothelial cell density was greater at 1 year in the dsaek group as compared with the penetrating keratoplasty group ( 2174 66 versus 1555 76 ; p = 0.001 ) and overall percentage of endothelial cell loss after 1 year was 40.9% 2.9% in penetrating keratoplasty - treated and 22.4% 2.3% in dsaek - treated eyes ( p < 0.001 ) . in patients with fuchs dystrophy , dsaek - treated eyes had significantly less percentage of endothelial cell loss compared with penetrating keratoplasty - treated eyes ( 30 eyes each group , mean percentage endothelial cell loss ; 20.4% 1.4% versus 37.7% 2.3% ; p = 0.001 ) .
this was similar to patients with bullous keratopathy ( 30 eyes each group , mean percentage of endothelial cell loss ; 24.2% 2.0% versus 41.8% 2.2% ; p = 0.002 ) .
we also compared percentage of endothelial cell loss at 1 year between patients with fuchs dystrophy and bullous keratopathy . at 1 year , the percentage of endothelial cell loss did not differ significantly between patients with fuchs dystrophy and those with bullous keratopathy for either procedure ( 39% versus 44% in penetrating keratoplasty - treated eyes ; p = 0.40 ; and 20% versus 24% in dsaek - treated eyes ; p = 0.45 ) .
in our study cohort , 26 patients had concomitant eye disease , which significantly impacted on final visual outcome , while 17 patients had late graft failure at 1 year .
all these patients were excluded from our final visual acuity analysis ( table 2 ) .
we compared the remaining patients ( 61 dsaek with 137 penetrating keratoplasty - treated eyes ) with respect to visual acuity . amongst these patients
there were fewer patients who had fuchs dystrophy in the penetrating keratoplasty group ( 35/61 [ 57.3% ] dsaek ; 51/137 [ 37.2% ] penetrating keratoplasty ; p = 0.011 ) .
patients who underwent dsaek had better visual outcomes at 1 year when compared with penetrating keratoplasty - treated patients ( table 3 ) .
overall , dsaek - treated eyes had significantly superior uncorrected visual acuity ( ucva ) and best spectacle - corrected visual acuity ( bscva ) as compared with penetrating keratoplasty - treated eyes at 1 year ( mean difference ucva , 0.42 0.0059 ; p < 0.001 ; bscva , 0.14 0.032 ; p < 0.001 ) .
in addition , there were more eyes with bscva better than 20/40 in dsaek - treated group as compared with the penetrating keratoplasty - treated group ( 67.2% versus 38.7% ; p < 0.001 ) .
when comparing visual outcomes between surgical indications , we found that visual outcome was significantly better in eyes with fuchs dystrophy for both dsaek ( mean difference bscva , 0.36 0.011 ; p = 0.002 ) and penetrating keratoplasty ( mean difference bscva , 0.40 0.0079 ; p < 0.001 ) .
overall , patients with penetrating keratoplasty - treated eyes had higher astigmatism at 1 year as compared with dsaek - treated eyes ( 3.0 2.1 versus 1.7 0.8 , respectively ; p < 0.001 , table 1 ) .
we also observed this difference in patients with fuchs dystrophy ( 2.6 1.8 versus 1.7 0.8 ; p = 0.023 ) and bullous keratopathy ( 3.2 2.2 versus 1.7 0.8 ; p = 0.001 , table 3 ) .
overall , dsaek - treated eyes were more hyperopic as compared with penetrating keratoplasty - treated eyes at 1 year ( spherical equivalent + 1.5 3.3 versus 0.25 1.8 , respectively ; p < 0.001 ) .
we had one case of primary graft failure in each of the dsaek and penetrating keratoplasty treatment groups ( 1/68 , 1.5% versus 1/173 , 0.5% respectively ; p = 0.31 ) . at 1 year , 66/68 ( 97.0% ) eyes that underwent dsaek had clear grafts while 158/173 ( 92.0% ) of penetrating keratoplasty - treated eyes had clear grafts ( p = 0.479 ) .
there were no significant differences in late graft failure between the groups ( penetrating keratoplasty 8% versus dsaek 3% ; p = 0.118 ) .
reasons for graft failure in the penetrating keratoplasty group were infection - related ( n = 10 ) and immune - related ( n = 5 ) ; and for the dsaek group were immune - related ( n = 2 ) .
the kaplan meier probability of survival at 1 year was 95.3% for the penetrating keratoplasty - treated group , which decreased to 89.6% at 18 months , while it was 98.4% at 1 year and 93.2% for dsaek cases up to 18 months of follow - up ( p < 0.001 ) .
complications during the 1-year follow - up period were recorded according to our singapore corneal transplant study guidelines.14 we scored complications as events and made comparisons between the penetrating keratoplasty and dsaek groups ( table 4).18 epitheliopathy was significantly higher in penetrating keratoplasty - treated eyes ( p = 0.014 ) , while transient episodes of intraocular pressure elevation > 21 mmhg ( defined in terms of short - term , ie , 3 months use of antiglaucoma medications ) were seen in 20/68 ( 29.4% ) and 52/173 ( 30.0% ) for dsaek and penetrating keratoplasty , respectively ( p = 0.93 ) . of note ,
most of these patients ( 16/20 [ 80.0% ] for dsaek and 49/52 [ 94.2% ] for penetrating keratoplasty ) had an underlying history of glaucoma ( p = 0.07 ) .
only four eyes ( three penetrating keratoplasty , one dsaek ) had had trabeculectomy performed previously , and there were no significant differences in graft outcomes .
all eyes with transiently raised intraocular pressure were treated successfully with intraocular pressure - lowering topical and/or systemic medications .
one patient who underwent dsaek had an acute graft rejection episode successfully treated with topical steroids , as compared with 11 ( 6.3% ) in the penetrating keratoplasty - treated group .
of note , we had no graft dislocations in our dsaek - treated eyes in this series of patients .
in this study we found that 1-year percentage of endothelial cell loss and visual outcomes were superior in dsaek - treated eyes as compared with penetrating keratoplasty - treated eyes from the same study cohort , which confirms the results of our preliminary study on dsaek in asian eyes.16 this is in contrast with reports in the literature that dsaek has greater endothelial cell loss compared with penetrating keratoplasty , but few of these studies reported data on endothelial cell loss beyond 12 months of follow - up and none were directly compared with penetrating keratoplasty from the same study cohort.3,812 one study compared subjects from their prospective trial on dsaek with a separate study on penetrating keratoplasty from the specular microscopy ancillary study cohort.19 another study found that 1-year endothelial cell density was lower with dsaek compared with penetrating keratoplasty albeit but the difference was not statistically significant.20 this led to the conclusion in a recent review article that there is currently insufficient evidence to conclude whether endothelial cell loss is greater in dsaek.21 the results from this study are encouraging because our endothelial cell loss at 1 year ( 22.4% 2.3% ) using a sheets glide nonfolding technique was comparable with other non - folding techniques , and we were able to show less endothelial cell loss comparable with penetrating keratoplasty from a same cohort of patients . although our endothelial cell loss is less than that using a taco - folding technique,22 longer follow - up is required to confirm if we have a similar endothelial cell loss trend .
another advantage of performing dsaek over penetrating keratoplasty is the ability to use a larger donor size , and thus transplant more endothelial cells.4 in our study , the mean donor size was 10% larger and endothelial cell density was greater in the dsaek group than in the penetrating keratoplasty group ( p < 0.001 ) .
the percentage endothelial cell loss , visual outcomes , and astigmatism in eyes with fuchs dystrophy and bullous keratopathy treated with penetrating keratoplasty in our study of asian eyes were comparable with those of previous reports in caucasian eyes.3,2325 however , it may not be useful to compare visual outcomes at 1 year between dsaek and penetrating keratoplasty due to the longer visual rehabilitation and use of sutures in penetrating keratoplasty , thus it was only a secondary outcome measure .
nonetheless , in our study , dsaek had better postoperative ucva and bscva as compared with penetrating keratoplasty , which was also found in caucasian eyes but with fewer eyes ( 20 in each group).3,20 while this is somewhat expected , our results confirm that visual rehabilitation takes much longer after penetrating keratoplasty , even when comparing bscva at 1 year follow - up . in our study cohort
there was a higher proportion of pseudophakic eyes amongst patients who had undergone dsaek for fuchs dystrophy .
this is because we adhere to the general policy that lens removal prior to dsaek obviates the risk of subsequent cataract formation and also creates more space in the anterior chamber during dsaek surgery .
moreover , more of the penetrating keratoplasty - treated eyes had complicated surgeries that required anterior vitrectomy and anterior chamber intraocular lens insertion .
these factors could contribute to the poorer visual outcome and lower endothelial cell counts in the penetrating keratoplasty - treated group .
the main limitations of our study are due to its retrospective nature . although our patients differed in their characteristics , such as demographics and phakic status , we matched subjects in each group for donor and recipient characteristics .
we acknowledge that case selection between dsaek and penetrating keratoplasty was dependent on surgeon choice , and that more severe or advanced cases of corneal decompensation were likely to have received penetrating keratoplasty surgery . however , we do routinely attempt dsaek in severe cases of bullous keratopathy , as long as there is only moderate anterior stromal scarring present .
this may have affected our postoperative visual outcome comparisons , although this would have minimal effect on differences in endothelial cell loss postoperatively between dsaek and penetrating keratoplasty .
our study also had a limited follow - up period of 1 year in order to compare graft survival and record reliable endothelial cell count data , which was only obtained in 120 eyes .
however , we analyzed eyes from each group matched for surgical indication to minimize selection bias and there were no significant baseline differences in each group
. nevertheless , due to the inherent success of dsaek , it would be challenging and unethical in our institution to conduct a randomized controlled study given the clinical advantages of dsaek over penetrating keratoplasty , hence a historical cohort of penetrating keratoplasty cases in the same population group using the same postoperative regime provided the best comparative cohort .
we found that performing dsaek using the described technique in asian eyes resulted in a lower 1-year endothelial percent cell loss as compared with penetrating keratoplasty for patients with fuchs dystrophy and bullous keratopathy .
graft survival was comparable in both groups at 1-year follow - up , although visual outcomes were superior in the dsaek group with fewer complications .
longer follow - up will provide more data on endothelial cell loss with these two techniques .
improvements in donor insertion devices may reduce the initial ecc loss further and improve long - term ecc outcomes.26 | backgroundthe purpose of this study was to compare endothelial cell counts after descemet s stripping automated endothelial keratoplasty ( dsaek ) and penetrating keratoplasty in asian eyes.methodsthis was a retrospective study of patients from our prospective singapore corneal transplant study cohort who received corneal transplantation in 20062008 .
we compared eyes that underwent dsaek or penetrating keratoplasty for fuchs endothelial dystrophy or pseudophakic and aphakic bullous keratopathy .
clinical data , and donor and recipient characteristics were recorded .
of 241 patients who met our inclusion criteria , 68 underwent dsaek and 173 underwent penetrating keratoplasty .
the main outcome measure was endothelial cell loss at 1 year .
secondary outcome measures were graft survival and visual outcomes at 1-year follow-up.resultsthere were no significant differences in baseline characteristics of patients between the treatment groups .
percent endothelial cell loss at 1-year follow - up was greater in penetrating keratoplasty eyes ( 40.9% 2.9% ) compared with dsaek eyes ( 22.4% 2.3% ; p < 0.001 ) .
dsaek - treated eyes had significantly superior uncorrected visual acuity ( mean difference = 0.42 0.0059 ; p < 0.001 ) and best spectacle - corrected visual acuity ( mean difference = 0.14 0.032 ; p < 0.001 ) as compared with penetrating keratoplasty - treated eyes .
penetrating keratoplasty - treated eyes had worse astigmatism as compared with dsaek - treated eyes ( 3.0 2.1 versus 1.7 0.8 ; p < 0.001 ) .
graft survival at 1 year was comparable in both groups , ie , 66/68 ( 97.0% ) dsaek - treated eyes versus 158/173 ( 92.0% ) of penetrating keratoplasty - treated eyes had clear grafts ( p = 0.479).conclusionwe report lower percent endothelial cell loss comparing dsaek and penetrating keratoplasty at 1-year follow - up in asian eyes , with comparable graft survival rates in both groups . |
pterygium is an ocular lesion that is usually benign and is frequent in sunny countries .
the intervention consists of the excision of the pterygium followed by the closing of the exeresis location .
excimer laser , beta irradiation , and antimitotic molecules can be associated with this surgery.1 infectious complications are rare .
the authors describe a case of keratomycosis that appeared after the exeresis of a pterygium .
a 48-year - old patient had been referred for a red right eye , associated with an abscess of the cornea along the ablation zone of the pterygium .
surgery had been performed a month beforehand and had involved the exeresis of a grade 2 recurring nasal pterygium .
the patient did not leave her house , except between days 8 and 15 post surgery where she spent some time outside in her garden . on day 25 post surgery , the increase of the secretions as well as
the appearance of a white area on the surgical site led the patient to consult her ophthalmologist .
the symptoms persisted and the patient was referred to the service 4 weeks post surgery .
there was a 6 mm high and 4 mm wide abscess of the cornea around the ablation site of the pterygium .
ulceration with raised edges of about 3 mm was at the center of the abscess .
the main abscess , along with several microabscesses , was located in the temporal region .
a discreet proteic tyndall was noted , as well as folds of the descemet s membrane of the nasal hemicornea .
there was no hypopyon or posterior synechia and no ocular hypertonia ( figure 1 ) .
a clinical aspect evoking a fungal keratitis and the revealing of several septate filaments on direct examination led the authors to establish a treatment involving ticarcillin ( 7 mg / ml ) , gentamicin ( 15 mg / ml ) , vancomycin ( 50 mg / ml ) , and amphotericin b ( 0.25% ) .
the antifungal treatment was prescribed as follows : one instillation every 5 minutes for 1 hour , then every hour for 3 days , then eight times a day for 2 months , and then , finally , six times a day for 1 month .
reinforced collyriums were replaced on day 8 by ciprofloxacin ( six times per day ) and this was maintained for 15 days .
cycloplegic collyrium was prescribed for 4 weeks and artificial tears for 12 weeks . on day 10 , a fusarium dimerum was isolated on the sabouraud agar . after 15 days of treatment
, the evolution was favorable and the size of both the ulceration and the abscess had progressively diminished .
the abscess was replaced with a whitish stromal scar with a big vertical axis and a diameter of about 5 mm ( figure 2 ) .
there was no more ulceration , but the cornea appeared slimmer within the ablation zone of the head of the pterygium .
surgery of the pterygium is usually considered as having a low rate of complications of infectious origin .
nevertheless , infectious complications can appear a few days or even a few years post surgery .
it essentially concerns bacterial agents ; pseudomonas aeruginosa is the germ that is usually the cause .
other infections by fungus are even more rare ; in a study by lin et al2 of 30 eyes infected after pterygium surgery , only one case of fungal origin ( cephalosporium ) was included .
other than cases reported by moriarty et al3 ( complications appeared 15 years after radiotherapy ) and peponis et al4 ( complications appeared 3 weeks post surgery and following use of mitomycin c ) , to the authors knowledge , this is the first case of a keratomycosis due to f. dimerum reported that complicated the exeresis of a pterygium without the use of an adjuvant anti - healing treatment . in this case , the evolution of the first observation was good , contrary to the second observation when the extension of the infection beyond the ocular structures led to the realization of enucleation .
like pterygium , keratomycosis is more frequent in tropical countries.5 the breaking of the corneal epithelium following surgery encouraged the appearance of a mycosis ; the gardening undertaken by the patient is the obvious source of a vegetal foreign body .
all instances of potential risk related to foreign bodies of any nature , including vegetal , must be avoided until complete re - epithelialization of the corneal ablation site of the pterygium .
although pterygium surgery is a commonly performed procedure , ophthalmologists should be aware of potential infectious complications that can have dramatic outcomes . | the authors describe a case of keratomycosis that appeared after the exeresis of a pterygium .
a 48-year - old patient had been referred with a red right eye associated with an abscess of the cornea along the ablation zone of the pterygium .
the surgery had been performed a month beforehand .
the abscess was 6 mm high and 4 mm wide .
the authors instigated a treatment that included amphotericin b ( 0.25% ) after noticing a clinical aspect evoking a fungal keratitis and finding several septate filaments on direct examination . on day 10 ,
a fusarium dimerum was isolated on sabouraud agar .
after 15 days of treatment , the result was favorable and the size of the ulceration as well as the size of the abscess had progressively decreased .
the antifungal treatment was definitively stopped at 14 weeks .
infectious - related complications of the pterygium surgery are rare and are essentially caused by bacterial agents .
secondary infections by fungus are rare .
there have been two previous cases reported : one that appeared 15 years after radiotherapy and another that appeared at 3 weeks post surgery , consecutive to the use of mitomycin c. to the authors knowledge , this is the first case of a keratomycosis due to f. dimerum reported that complicated the exeresis of a pterygium without the use of an adjuvant antihealing treatment .
pterygium surgery is a common procedure ; nevertheless , ophthalmologists need to be aware of the existence of potential infectious complications . |
although the statistical - mechanical
foundations of free - energy
calculations were laid a long time ago , their practical applications
became possible only with the advent of modern computers . from the
inception of computer - based free - energy calculations it has been clear to theorists that direct boltzmann sampling of
rugged energy landscapes is inefficient .
the subsequent development
of the field is a history of efforts to remedy this problem . in free - energy calculations ,
the quantity of interest is almost
always the free - energy difference between physical
states of the system rather than the absolute free energy of a given
state . from this standpoint
, calculations can be categorized on the
basis of variables used to transform the system between states of
interest .
then , two main classes can be distinguished , namely alchemical
and geometrical transformations .
they
rely , respectively , on changes of a parameter in the hamiltonian or
a function of atomic coordinates .
the first class encompasses structural
modifications of chemical species that rest upon the remarkable malleability
of the potential energy function in molecular - mechanics - based simulations , reminiscent of the fabled ability of alchemists to transmute base
metals into noble ones .
alchemical transformations are often associated
with the free - energy perturbation method on account
of the progressive and perturbative nature of the change incurred
by the system of interest , although , strictly speaking , alchemical
free - energy calculations can be carried out by way of alternate approaches ,
such as thermodynamic integration .
the
very first application of alchemical transformations to a nontrivial
chemical problem was published nearly 30 years ago by william jorgensen ,
to whom the present contribution is dedicated . in noteworthy agreement with experiment ,
this pioneering simulation
reproduced the relative hydration free energy of methanol with respect
to ethane .
the second class of transformations embraces virtually
any geometric
modification in a molecule or a collection of molecules by means of
selected collective variables tailored to address the problem at hand ,
which could vary from changes in the internal degrees of freedom in
a molecule to intricate recognition and association phenomena .
such collective variables form the transition
coordinate , a low - dimensional representation of a multidimensional
mathematical object .
the distinction between the two types of
transformations is theoretically
important . for geometric transformations ,
the transition coordinate
is , in effect , a generalized coordinate , the evolution of which is
usually described by hamilton s equations of motion .
in contrast ,
for a parameter in the hamiltonian , no equations of motion naturally
exist , although it is possible to extend the formalism of dynamics
to include such a parameter . as a consequence
,
a number of methods for calculating free energy by way of geometric
transformations can not be applied to alchemical transformations without
such extension .
a considerable number of
ingenious techniques have been developed
to improve the efficiency of mapping free - energy landscapes associated
with geometrical transformations along a transition coordinate .
a common feature of these methods is their reliance on importance - sampling
techniques .
the central idea of these techniques is to depart from
sampling from the boltzmann distribution defined by the original hamiltonian
and , instead , sample from another distribution that favors regions
of phase space that would be visited only infrequently but are important
to achieving reliable free - energy estimates .
because this procedure
is clearly biased , it is essential to know how to correct , or unbias ,
it to recover the true underlying distribution .
importance sampling
is commonly used not only in statistical mechanics of condensed phases
but also in other fields of science , usually as a variance reduction
technique most frequently combined with the monte carlo method .
probably the most popular , and also the oldest importance - sampling
technique used in free - energy calculations is umbrella sampling .
it relies on introducing a bias in simulations
that favors states corresponding to large values of the free energy
along the transition coordinate . local elevation , conformational flooding , metadynamics , and the wang
landau algorithm are examples
of more recent importance - sampling algorithms , united by the common
denominator that a memory - dependent potential disfavors regions of
conformational space that have already been frequently visited . in a sense ,
the adaptive biasing force method rewove the fabric of free - energy calculations of geometrical
transformations , as it is characterized by both conceptual and practical
simplicity and requires , at least in principle , little intervention
from the end user . in spite of apparent similarities with the local - elevation
and conformational - flooding strategies in its aim to sample efficiently
all values of the transition coordinate , its theoretical underpinnings
are quite different , as we will argue further in this paper .
furthermore ,
in contrast with seemingly similar strategies , the adaptive biasing
force algorithm requires no prior knowledge of the free - energy landscape
at hand . at its core ,
the adaptive biasing force method is an
adaptive importance - sampling
strategy in which the quantity being adjusted is the average force
acting along the transition coordinate .
it helps the system under
study escape from kinetic traps in which it would otherwise have remained
for a very long time .
the method constitutes a highly efficient route
to estimating free energies , which , since its inception , has been
used to tackle a number of challenging problems of chemical and biological
interest , such as mechanical proteins , transport phenomena , or protein ligand and protein protein
recognition and association . more generally ,
it is a versatile , adaptive , importance - sampling strategy that can
be utilized in many fields , whenever sampling of a probability measure
is thwarted by metastability of the sampling dynamics . in this
self - contained contribution ,
the multiple facets of the
adaptive biasing force algorithm are discussed in an exhaustive manner ,
tackling a number of issues that have not been addressed so far , or
only rarely so . in the following section
, we present the theoretical
foundations of the method , discussed in the context of other free - energy
approaches .
next , we briefly address a number of practical issues
related to a proper choice of the transition coordinate .
then , an
analysis of the convergence properties of the method and approaches
to calculating and controlling statistical errors associated with
the calculated free - energy values are presented .
the discussion of
convergence and errors continues with the focus on nonergodicity scenarios ,
and ways to identify and circumvent them .
subsequently , we examine
for the first time how the adaptive biasing force algorithm is used
in conjunction with geometrical restraints , which have to be enforced
in many problems of interest . finally , we discuss some new strategies
for combining thermodynamics and kinetics in importance - sampling simulations ,
before closing with recommendations for good practices
in applying the adaptive biasing force method and an outlook toward
further promising statistical mechanical and algorithmic developments .
in this section , the essential
idea behind the adaptive biasing
force algorithm is first explained in terms appealing to physical
intuition , followed by the theoretical underpinnings of the method
presented in a more formal language .
then , the reader is guided through
the common expressions for the mean force , and the adaptive algorithm ,
both of which are at the core of the method .
the adaptive
biasing force method is aimed at improving the efficiency of molecular
dynamics simulations in which the potential energy surface is sampled
ineffectively due to free - energy barriers . in practice , these barriers
appear as bottlenecks in the dynamics of certain privileged coordinates
that describe the transitions between physically important states
( transition coordinates ) .
they also cause the system to become trapped
in some states for durations exceeding the time scale of the simulation ,
resulting in incomplete sampling .
the free energy along a transition
coordinate can be seen as a potential resulting from the average force
acting along the coordinate ( i.e. , the negative of the gradient of
this potential ) , hence the name potential of mean force . in the formalism of thermodynamic integration ,
on which the adaptive biasing force is based ,
the instantaneous force acting
along the coordinate may be decomposed into the sum of the average
force ( which depends only on the value of the transition coordinate )
and a random force with zero average , reflecting fluctuations of all
other degrees of freedom .
hence , in a low - dimensional view of the
process , the transition coordinate evolves dynamically in its time - independent
potential of mean force , and this evolution is driven by the random
force . in many instances ,
the random force can be satisfactorily approximated
as diffusive , leading to a simple physical picture in which the system
diffuses along the transition coordinate in the potential of mean
force .
the idea behind the adaptive biasing force algorithm
is to preserve
most characteristics of this dynamics , including the random fluctuating
force , while flattening the potential of mean force to remove free - energy
barriers , and thus accelerate transitions between states .
this is
done adaptively , without any prior information about the potential
of mean force . to accomplish this ,
the instantaneous force acting
along the coordinate is calculated , and its running time average is
recorded , thus providing an on - the - fly estimate of the derivative
of the free energy at each point along the pathway . at the same time ,
an external biasing force is applied , exactly canceling the current
estimate of the average force . over time , as the estimate converges
to the average force at equilibrium , the total , biased average force
stabilizes at values very close to zero . then , the system experiences
a nearly flat potential of mean force and displays accelerated dynamics
along the transition coordinate .
the fact that the biasing force is
exactly equal to the mean force is actually not crucial .
what is important
is that the biasing force yields sufficiently uniform sampling of
the transition coordinate that the remaining barriers can be easily
traversed in response to thermal fluctuations .
the adaptive biasing force
algorithm is not inherently tied to any specific type of dynamics
but does rely on sampling of the canonical ensemble . in explicit - solvent
simulations , langevin dynamics with sufficiently soft damping and
small stochastic forces becomes a mere perturbation of hamiltonian
dynamics and may be used as one simple way to achieve canonical sampling .
for convenience , but without loss of generality
langevin dynamics can be written
as1where
( xt , pt ) denotes the positions and
momenta of the particles at time t , m is the mass tensor , v : is the potential energy function ,
is the friction coefficient , wt is the wiener process that underlies the random force ( white
noise ) , and = kbt , where kb is the boltzmann
constant and t is temperature .
the dynamics of eq 1 is ergodic ( under mild conditions on v ) with respect to the canonical measure zp1 exp[pmp/2 ] dp (dx ) , where (dx ) = zx1 exp[v(x ) ] dx .
ergodicity means
that long - time averages converge to canonical averages:2and that the law at time t of the stochastic process converges to the canonical measure
in
the long - time limit:3where is
the expected value .
the first limit
in eq 2 is of particular interest for practical
applications because it allows for computing canonical averages from
trajectory averages . for a typical potential
v , the associated boltzmann measure is multimodal : high - probability
regions are separated by low - probability regions .
the former correspond ,
for instance , to the most likely conformations of a biological object ,
which are typically separated by transition regions of very low probability .
for these reasons , estimating averages with respect to the probability
measure is , in general , a difficult task . in particular , the
ergodic properties of the dynamics of eq 1 are
not sufficient to devise reliable numerical methods , because under
these premises , the stochastic process remains trapped in large - probability
regions , and , as a consequence , the long - time asymptotic regime t is very difficult to reach in the ergodic
limits in eqs 2 and 3 .
the fact that the system remains for a very long time in some region
of phase space before hopping to another region is called metastability ,
and the corresponding states of the system are called metastable .
the inability to reach the ergodic limit
is often called quasi nonergodicity ;
the system appears nonergodic on the time scales of the simulations .
a typical example of a metastable state is a local free - energy minimum
in the conformational space of a protein .
the adaptive biasing
force method relies on modifying the potential v in
such a way that the energy landscape is flattened along
a given transition coordinate : . here ,
we restrict ourselves to a one - dimensional
transition coordinate , leaving generalization to high - dimensional
transition coordinates for the section expressions
for the mean force .
at[(x ) ] and at is
updated in such a way that it converges to the free energy a , defined ( up to an additive constant ) by4where the measure (x)z ( dx ) is supported by the subset
{ x , (x ) = z } and
is such that (x)z ( dx ) dz = dx . in practice , the bias is only applied in a window [ zmin , zmax ] as explained
in
the section justification of a stratification strategy .
notice that , by the definition of a , the canonical
measure associated with the biased potential v(x )
= c , where c is a constant
independent of z. therefore , if the biased potential v(x ) a[(x)]1(x)[zmin , zmax ] is
used , the marginal along is a uniform
law over [ zmin , zmax ] . here , 1(x)[zmin , zmax ] is
an indicator function , which is one when (x )
[ zmin , zmax ] and zero otherwise . let us recall that the marginal law is defined
as follows : if x is distributed according to a probability
distribution , then the law of (x ) is called
the marginal of along . if we knew the free energy a , it would be a good idea to use a as a biasing potential , because sampling along
would be easier .
this is illustrated in simple two - dimensional examples
in figures 1 and 2 .
notice in particular that the free energy seems to be a good biasing
function for efficient sampling of both energetic barriers ( figure 1 ) and entropic barriers ( figure 2 ) .
upper row : ( a ) original two - dimensional , double - well potential
displayed as level sets ; ( c ) time trajectory of the first coordinate x in the stochastic process , showing oscillations between
the two metastable wells .
lower row : ( b ) level sets of the same potential
biased by the free energy associated with the transition coordinate
(x , y ) = x ; ( d ) time trajectory of the transition coordinate x in the adaptively biased dynamics , showing no metastability .
upper row : ( a ) the original two - dimensional
potential is zero inside
the hourglass shape , and + outside ; ( c ) time trajectory of
the first coordinate x in the stochastic process ,
showing oscillations between the two metastable wells .
lower row :
( b ) free energy along the transition coordinate (x , y ) = x , featuring a purely entropic
barrier ; ( d ) time trajectory of the transition coordinate x in the free - energy - biased dynamics , showing no metastability .
the main ingredient that we now
need is an update rule for at such that limtat = a. this is based on the following formula , which defines
the mean force , namely , the negative of the derivative
of the free energy .
more general formulas can be derived , which give
rise to many variants of the adaptive biasing force method ( see section expressions for the mean force),5where the instantaneous force is defined by6 equation 5 is a consequence of the definition
of the free energy in eq 4 and of the co - area
formula ( which is a generalization of the fubini theorem ) ; see for
instance ref ( 8) . from
eq 5 it follows that a
is the conditional average of the instantaneous force , f , with respect to the canonical measure conditioned
to a fixed value of the transition coordinate : a(z ) = [ f(x)(x ) = z ] .
an important observation is that
eq 5 remains
true if the potential v is changed to v
at :
for any function at7 in other words , a biasing potential at depending solely on leaves averages
conditioned
by unchanged .
this is the intuition behind the adaptive biasing
force dynamics , which can be written as8 indeed , if ( xt , pt ) were at equilibrium with respect
to the biased canonical measure zp1 exp(pmp/2 ) dpzt1 exp[(v
at)(x ) ] dx , then at would be equal to a. in practice ,
of course , the sampling is not sufficiently fast for the process to
be instantaneously at equilibrium with respect to the time - varying
biased potential v
at , and this is why this heuristic
is not sufficient to fully understand the convergence of the adaptive
biasing force dynamics ( see the section convergence
and error analysis ) . however , this simple reasoning is sufficient
to check that if at converges
to some limit a , then necessarily , a = a. from a practical
viewpoint , the conditional expectation in eq 8 can be computed using two procedures : either time
averages over a single long trajectory or averages over many replicas
run in parallel .
these procedures will be discussed in ample detail
in the section multiple - walker strategies below . intuitively , the adaptive biasing force dynamics thus
consists
of adding a force at[(x)](x ) that exactly compensates the average of the original force ,
v(x ) , along a given
transition coordinate .
if is well - chosen , the hope is to observe
a fast convergence ( at least compared to the original dynamics embodied
in eq 1 at equilibrium ) of at to the mean force a. given a transition coordinate
(x ) , the mean force is a well - defined quantity ,
yet its expression as an ensemble average of an instantaneous force f is not unique , as we will see below .
in adaptive biasing force simulations ,
the choice of a convenient
expression for f is driven by practical
considerations , notably ease of implementation and numerical behavior ,
such as variance . the classic expression for
the instantaneous
force involves an explicit coordinate transformation from
cartesian to generalized coordinates , which include the transition
coordinate . that is , : , with 1 = and
components i for i > 1 are generalized coordinates of no particular physical significance ,
but necessary to the mathematical framework .
a valid expression for
the instantaneous force is then9which in the physics literature is
more commonly
written as10where |j| is the determinant
of the jacobian matrix ( ij1)(i , j ) . from the arbitrary choice of
i , i>1 is derived
a ( somewhat arbitrary ) function f , whose -restricted ensemble average nevertheless yields the
uniquely defined mean force ( eq 5 ) . equation 10 provides an intuitive view that different choices
of and f correspond to
different ways of projecting the cartesian forces , v , onto the transition coordinate .
the direction along which
the forces are projected in this expression is the vector 1 , which we call inverse
gradient . as the gradient of
can be seen as the changes in corresponding to infinitesimal
changes in x , the inverse gradient is the vector along
which a change in is propagated in cartesian coordinates ,
other generalized coordinates ( i , i>1 ) being constant , hence the dependence of the
inverse
gradient on the explicit coordinate transformation .
the alternate
notation for the inverse gradient , x/ ,
has the drawback of hiding this dependence on the choice of . numerically , eq 10 is impractical for two
reasons .
one is that defining i , i>1 explicitly can be exceedingly difficult , especially
if is a collective coordinate ( e.g. , the radius of gyration
of a set of particles ) .
supposing that this step is done , the second
difficulty comes with the numerical computation of the jacobian derivative ,
as it involves second derivatives of whose
analytical derivation and numerical implementation may be cumbersome ,
again , depending on the nature of the transition coordinate . to circumvent this issue ,
the original adaptive biasing force method
was introduced with an instantaneous force estimator based on a constraint
force that is calculated iteratively , but never applied . in the initial implementation of the adaptive
biasing force algorithm in the popular
molecular dynamics program namd , eq 10 was used because the small set of implemented coordinates
made it practical .
as this set was greatly extended in the framework
of the collective variables module , more
versatile expressions of the instantaneous force were required .
den otter put forward the idea that the
inverse gradient can be
replaced with an arbitrary vector field ( satisfying certain requirements ) . in other words ,
changes in may be propagated
along an arbitrary direction in cartesian coordinates , without explicitly
defining a complete set of generalized coordinates .
consider a vector transition coordinate ( i ) , in the presence of a set of constraints
of the form k(x ) =
0 .
for each coordinate i , let vi be a vector field satisfying , for all j and k:1112 the ith partial derivative of the free energy
can then be calculated as the conditional average of the following
instantaneous force:13of which
eq 6 is a special
case , limited to a single coordinate and choosing v =
/|| , which satisfies the
condition ( eq 11 ) above .
note that this estimator
still requires the calculation of second derivatives , in the form
of the divergence of the vector field v , although the
relative freedom in choosing v can be taken advantage
of to make the divergence calculation practical .
the choice v = /|| is always
valid , and as such , convenient for theoretical purposes , but certainly
not always optimal when implementing specific generalized coordinates .
expressions that were chosen in practice for those coordinates implemented
in the collective variables module are listed in ref ( 39 ) .
described
an estimator that does not require second
derivatives , but rather first derivatives with respect to time and
space , and is valid for multidimensional adaptive biasing force calculations .
this estimator resembles a statistical form
of newton s equation of motion : instead of relating acceleration
to the potential energy gradient , it relates the mean acceleration
to the gradient of the free energy . in a considerable simplification ,
only the first derivative of the force with respect to needs
to be derived .
the time derivative is calculated numerically in the
same fashion and at the same level of accuracy as time derivatives
of other quantities in molecular dynamics .
other ways of simplifying
calculations of instantaneous forces will be discussed in the context
of extended adaptive biasing force simulations , or eabf , in the section the extended adaptive biasing force method .
the final , essential ingredient
of the theory underlying the adaptive force method is an algorithm
for deriving the current estimate of the average force as simulations
progress . in its generic , one - dimensional implementation , the transition
coordinate , , connecting two end points ,
is divided into m equally sized bins of width in which forces are accrued in the course of the simulation . in
a nave approach ,
the approximation to the average force , f(nstep , k ) in bin k after nstep molecular dynamics steps is just the simple , unweighted
average of all force samples in this bin14provided that the bin has already been visited
at least once .
nstepk is the number of samples
accrued in bin k after nstep steps and fk abbreviates the th
force sample in this bin .
however , when only a few samples are available
in a given bin k , the running average might be a
poor estimate of the actual average force in this bin .
large fluctuations in the running estimate of the average force
are undesirable , as they may drive the system away from equilibrium ,
thus slowing the convergence of the algorithm and reducing the efficiency
of the method . to control these effects , a procedure is needed to
reduce variations in early estimates of f(nstep , k ) .
a number of schemes can be applied for this purpose . in current
implementations , the biasing force in bin k at time
t is applied in full only if the number of samples ntk is above a threshold , nfull .
it is ramped up smoothly as ntk varies from 0 to nfull . in one implementation ,
the
ramp is linear and the force is proportional to ntk / nfull ; in another , the biasing force is zero for ntk < nfull/2 and is ramped linearly
above that value , proportionally to 2ntk / nfull 1 .
both
implementations have proven to be efficient in a number of applications ,
but other , more advanced schemes are possible .
so far , there have
been no systematic studies on the efficiency of different adaptive
algorithms , but it is anticipated that it may be strongly system - dependent . for a sufficiently large nstep , f(nstep , k ) approaches the correct average force in each
bin .
then , the free - energy difference , a , between the end point states can be estimated simply
by way of summing the force estimates in individual bins.15if the average force is
a rapidly changing
function of , a more sophisticated integration algorithm may
be required
. it might be also possible to develop binless integration
algorithms , similar to those proposed for some other free - energy calculation
methods .
a common trait of importance - sampling
algorithms is the discretization
of the transition coordinate , , in bins of width in which statistical information is accrued in the course of the
simulation . in the umbrella - sampling scheme , for instance ,
a histogram is constructed , corresponding
to the biased probability of occurrence of the molecular assembly
of interest at the different values of the transition coordinate .
in the adaptive biasing force algorithm ,
bins are utilized to store instantaneous values of the thermodynamic
force that acts along the transition coordinate . as has been observed
in practice previously for diffusive dynamics
, the instantaneous force
in any given bin obeys a normal distribution . at thermodynamic equilibrium , by definition ,
its average is exactly
equal to a(z ) , that
is , the gradient of the free energy along the transition coordinate .
insisting upon being at thermodynamic equilibrium is pivotal here , as application of a poorly estimated time - dependent
bias , i.e. , from a distribution out of equilibrium
, will not yield
a hamiltonian bereft of a mean force acting along the transition coordinate .
the adaptive algorithms described above contain two adjustable
parameters : bin width , , and the number
of samples , ninit , below which r(nstepk ) is not equal to 1 or , equivalently ,
averaging does not follow eq 14 .
the choice
of these parameters should be done with some care . at constant , large values of ninit yield better estimates of the average force
once the number of samples collected in a given bin
reaches this threshold
value and conventional averaging begins , at the price of delayed application
of the full averaging and slow , initial progress along the transition
coordinate .
small values of ninit , in
turn , tend to drive the system out of equilibrium . typically , setting ninit in the range between 200 and 500 appears
to be a good compromise that allows for avoiding both types of problems . at constant ninit , large values of
this may have adverse effects on the
accuracy of integration in eq 15 . on the other
hand ,
small values of require longer
simulation times to collect sufficient force statistics in every bin .
if the transition coordinate is a distance in an atomistic system ,
the choice of as 0.1 or 0.2
usually represents a satisfactory trade - off . in the last four decades
a
number of strategies have been developed
for computing free - energy changes as a function of geometrical transformations ,
each endowed with advantages , as well as limitations . to a large extent ,
umbrella sampling ,
whether in its original formulation or variants thereof , remains one of the most widely
utilized routes to address rare events in molecular simulations . in
its original form ,
umbrella sampling referred to incorporating an
external biasing potential in the simulation , i.e. , an umbrella , ideally the negative of the free energy , which
would yield a broad , if not uniform exploration of the transition
coordinate .
in other words , in ideal circumstances , the system would
evolve in the collective - variable space on a flat free - energy hyperplane ,
as is also the case for the adaptive biasing force method .
under most
circumstances , however , the form of the optimal umbrella is unknown .
thus , for any qualitatively new problem , the end - user must resort
to an educated guess regarding the shape of the biasing umbrella potential ,
usually on the basis of prior knowledge of this and related problems .
this may constitute a daunting task . poorly
predicted biasing potentials yield nonuniform probability distributions
across the transition coordinate .
this decreases the efficiency of
umbrella sampling , which , in extreme cases , may reduce rather than
improve the efficiency compared to results with unbiased calculations .
this common shortcoming led to the development of an adaptive variant
of the umbrella - sampling algorithm , wherein
the initial guess of the biasing potential is progressively refined
in light of a series of short simulations .
adaptive umbrella
sampling is one member of a broader family of techniques called adaptive
biasing potential methods .
local elevation , conformational flooding , or its more recent avatar , metadynamics , adaptive biasing molecular dynamics , and the wang
the idea is to penalize the
already visited states by changing the potential v to v
at , at(z ) being related to the occupation time of the
value z of the transition coordinate up to time t. the longer the time spent in a bin { x , (x ) [ z0 , z1 ] } , the larger the biasing potential at(z ) , z [ z0 , z1 ] .
the biasing potential at is built as a sum of gaussian kernels that are periodically
added to the hamiltonian along the variable .
this pushes the
system away from states that have already been visited and , by doing
so , improves sampling .
it ought to be noted that the potential at , rather than its derivative ,
is computed in these two cases , hence , the name adaptive biasing potential
methods . one important downside of the adaptive biasing force algorithm ,
compared to the class of adaptive biasing potential methods , lies
in its inability to handle discrete transition coordinates , for instance ,
coordination numbers .
this drawback can be understood by considering
that the free energy is now a map from integers to reals , and , thus ,
has no derivative and , hence , no mean force . from a mathematical
viewpoint ,
the adaptive biasing force method ,
just like adaptive biasing potential methods , is an adaptive importance - sampling
procedure .
there is , however , a salient difference between these two
techniques . in the latter , the potential of mean force or , equivalently ,
the corresponding probability distribution along the transition coordinate
is being adapted .
in contrast , the former relies on biasing the force ,
i.e. , the gradient of the potential .
this difference is more important
than it might appear at first sight , as potentials and probability
distributions are global properties whereas gradients are defined
locally . in terms of probability distributions , it means that the
count of samples in the neighborhood of a given value of the transition
coordinate is insufficient to estimate probability .
knowledge of the
underlying probability distribution over a much broader range of
is required .
this may considerably impede efficient adaptation . in
contrast , all that is needed to estimate
the gradient is the knowledge
of local behavior of the potential of mean force .
thus , in many
instances , adaptation proceeds markedly faster . using a common metaphor ,
the difference between the adaptive biasing potential and adaptive
biasing force methods can be compared to inundating the valleys of
the free - energy landscape as opposed to plowing over its barriers
to yield an approximately flat terrain , conducive to unhampered diffusion .
for example , in metadynamics and its ancestors ,
the widths and weights of gaussian functions and the frequency with
which the biasing potential is updated have to be carefully chosen ,
which often requires considerable experience . in the adaptive biasing
force method , estimating the biasing gradient happens automatically
by way of a simple algorithm , described in the previous subsection .
another technical concern about adaptive methods is to ensure that
adaptation vanishes once at approaches the converged free energy .
there are a number of ways to fulfill this condition more - or - less
automatically , but adaptive biasing force and
adaptive biasing potential techniques remain intrinsically different
from this point of view . in the adaptive biasing force algorithm ,
if the correct free energy is given as an initial guess ( namely if v is replaced by v
a in eq 8) , then the biasing
force will not be updated ( at in eq 8 will be constant over time ) .
moreover , if the derivative
of the biasing potential is needed ( for example to bias the langevin
dynamics as in eq 8) , the advantage of the adaptive
biasing force algorithm is that at is directly
computed , whereas in adaptive biasing potential algorithms , one needs
to differentiate the evaluated biasing potential at , which may lead to very noisy results
because at is estimated
along a stochastic trajectory .
because the basic quantity calculated ,
and subsequently integrated ,
in the adaptive biasing force method is the force , this approach belongs
to the thermodynamic integration class of methods .
however , in contrast
to conventional implementations of thermodynamic integration and its
generalizations , such as the blue - moon ensemble approach , the adaptive biasing force algorithm does not
rely on constrained molecular dynamics , but instead is based on unconstrained
simulations ; i.e. , the free - energy difference is not determined at
discrete values of the transition coordinate through solving constrained
equations of motion . sampling of the transition pathway proceeds in
a continuous , unhampered fashion , guided by the diffusion properties
of the system of interest , obviating the need for re - equilibration
at fixed , predefined values of the transition coordinate , even in
stratified simulations . as will be discussed further in this paper
it is also
of interest to compare the adaptive biasing force method
with reconstructions of free - energy landscapes from nonequilibrium
trajectories that represent repeated pulling experiments .
the latter are based on the groundbreaking jarzynski identity , or its extension to bidirectional transformations , combined with steered molecular dynamics .
even
though both approaches involve molecular dynamics trajectories that
are initially away from equilibrium , there is a fundamental distinction
between them . in the adaptive force method , only the average , or systematic
force
is removed to erase the ruggedness of the free - energy landscape ,
preserving the random force responsible for diffusion .
once a good
estimate of the average force becomes available , the equilibrium behavior
of the system is restored . in pulling experiments , the transformation
always proceeds away from equilibrium at constant velocity and , therefore ,
the instantaneous force acting along the transition coordinate is
nil .
the random force actually appears in the formalism after averaging
over the ensemble of pulling experiments .
moreover , achieving convergence
in calculations based on jarzynski s identity usually requires
large numbers of independent realizations , which comes at a significant computational cost .
taken together ,
when a geometrical transformation can be undertaken at equilibrium ,
it is not clear whether there is any practical advantage of handling
the problem at hand by means of nonequilibrium work experiments rather
than the adaptive biasing force method .
an important advantage
of gradient - based methods is
the possibility of formally decomposing the free - energy change into
physically meaningful contributions , thereby helping
to dissect qualitatively the nature of the intermolecular interactions
at play .
it is worth noting that different energy terms contribute
to the mean force both explicitly through force terms and implicitly
through the boltzmann weights in the canonical average ; contributions
can only be separated numerically at the former level , not at the
latter .
decomposition of the free energy is generally handled a posteriori
through computing the thermodynamic force between , for instance , groups
of atoms of interest .
this force is then projected onto the transition
coordinate determined for each stored configuration , prior to the
construction of a histogram from which the average force is inferred .
integration of the latter yields the desired contribution to the total
free - energy change across the entire transition pathway . among
many options for reconstructing free - energy landscapes along
a transition coordinate , which one is the best ? considering that the
efficiency of different methods strongly depends on their implementation
in software packages and , very likely , on a system of interest , attempts
to answer this question appear somewhat misguided and unproductive .
that said , one aspect of the adaptive biasing force algorithm pleading
in its favor is its simplicity .
how the algorithm operates
is physically intuitive , requiring , in principle ,
very little prior knowledge of the free - energy landscape , or input
from the end - user , even for qualitatively new problems .
central to geometric transformations is the concept
of a transition
coordinate . in this section
, this concept is illuminated in the context
of free - energy calculations aimed at tackling rare events .
specifically ,
we will discuss how the transition coordinate is explored with the
adaptive biasing force algorithm and delve into the practical aspects
of defining this coordinate .
stratification , a common technique for improving the efficiency of free - energy calculations
by partitioning the reaction pathway into ranges of the transition
coordinate , will be discussed with the focus on the justification
for and limitations of this strategy . for any transition
from a macrostate , a , to another macrostate ,
b , of the same system
there exists an exact one - dimensional transition
coordinate : the committor probability . in most cases , this coordinate
is difficult to calculate and usually offers very limited insight
into the nature of the process of interest . for these reasons , it
is often more useful to employ a transition coordinate that is only
an approximation to the committor probability but is physically more
meaningful and easier to handle
. sometimes it might be helpful to
extend the reduced representation of the transition to a transition
coordinate that extends beyond one dimension .
not only physical significance
but also efficiency of sampling , given a transition coordinate , is
of concern .
as
in any enhanced sampling method based on a reduced representation ,
adaptive biasing force sampling relies on stochastic exploration along
the transition coordinate , , enhanced by the adaptive bias ,
combined with equilibration of other , orthogonal degrees of freedom .
equilibration in the orthogonal space is critical in two respects :
it affects the mobility along ( see figure 1b for a diffusive example ) , and it determines the rate of
convergence of a(z ) = f(x) , which is an average over the orthogonal degrees of freedom . in
the ideal situation of time scale separation , all slow degrees of
freedom are captured by the transition coordinates , and relaxation
in the orthogonal space is comparatively fast . in other words ,
the
adaptive biasing force algorithm removes metastability along the transition
coordinates , provided that other degrees of freedom are not metastable .
complex systems such as biological macromolecules , however , possess
many slow , coupled degrees of freedom , making time scale separation
difficult or impossible to achieve .
fortunately , empirical results
suggest that time scale separation
is not an absolute requirement of adaptive biasing force sampling
.
one reason behind it might be that enhanced diffusion in the transition
coordinate space reduces metastability in the orthogonal space , by
letting the dynamics sidestep orthogonal barriers
rather than cross them , making some multichannel
cases
( see the section hidden barriers and other challenges
to obtaining accurate results ) tractable with standard adaptive
biasing force simulations .
more encouraging still , convergence in
such multichannel cases can be markedly accelerated by multiple - walker
formulations of the adaptive biasing force algorithm ( see the section multiple - walker strategies ) . yet
as will be extensively discussed further in this paper , the end - to - end
distance of the -helical deca - alanine peptide provides a good
example of an inadequate transition coordinate . depending on the range
of values
, the coordinate exhibits completely different behavior .
for values corresponding to the stable -helix
( 14 ) and
larger , separation of time scales is obeyed and the adaptive biasing
force converges well .
in contrast , smaller
values of the end - to - end distance correspond to a rich set of metastable ,
compact states that are not resolved by the transition coordinate . as a result ,
adaptive biasing force dynamics
becomes trapped in these states , and the free - energy estimator does
not converge in accessible simulation times .
attempts to resolve these metastable states by two- and three - dimensional
coordinates give improved results , allowing the exploration of all
metastable basins , yet those basins are not all resolved even in three
dimensions .
these difficulties
are not due to specific deficiencies of the adaptive biasing force
method , but rather , to shortcomings of the reduced representation ,
which would constitute an obstacle to any sampling method . in practice ,
finding an effective reduced representation is still a process largely
guided by physical intuition about the process of interest , as well
as trial - and - error .
more systematic and robust approaches for this
dimension reduction step are an area of active research .
a limiting factor is often the availability
of usable numerical implementations of the generalized coordinates
of choice .
the collective variables module is an attempt to overcome this limitation , by providing a rich and
flexible toolbox to define many types of coordinates , in particular
those useful for the description of biological macromolecules . in
this module ,
once an intuitive understanding of the relevant
generalized coordinate has been obtained , some technical choices remain
to be made to express this coordinate as a function of atomic
cartesian coordinates .
though these decisions may seem ancillary ,
they have a strong influence on the accuracy , convergence , and computational
performance of the adaptive biasing force algorithm . when objects
of interest are composed of many atoms , there are
often several nearly equivalent ways to define the transition coordinate .
in sufficiently long simulations ,
different definitions produce nearly
identical potentials of mean force , but the efficiency might vary
considerably .
for example , the distance between two proteins could
be defined by selecting one central atom in each protein , or by selecting
the centers of mass of large groups of atoms .
the largest contribution
to the instantaneous force on each atom in a molecule is due to rapidly
oscillating bonded interactions ; more generally , in all applications ,
forces on the particles will contain some background noise . if many
atoms contribute to the projected force ( e.g. , the first right - hand - side
term in eq 13 ) , contributions from those noisy
terms average out , which lowers the variance of the instantaneous
force estimator . in the initial stage of an adaptive biasing
force simulation , nonequilibrium
effects occur if the biasing force applied to some degrees of freedom
varies faster than coupled degrees of freedom can relax .
collective variables
that involve many cartesian coordinates with smoothly varying contributions
to the gradient ( hence , to the biasing force vector ) . at equilibrium
,
a smooth motion may be described by a nonsmooth variable and this
may make the convergence of the adaptive bias more difficult . in the
deca - alanine stretching toy example , the geometric process of interest
involves all atoms in the peptide , yet our classic approach uses the
end - to - end distance as a biasing coordinate , with the implicit assumption
that biasing forces exerted on the terminal atoms propagate , and that
the entire peptide relaxes rapidly , so that the biased trajectory
remains close to equilibrium .
a more robust approach is to replace
that coordinate with the radius of gyration of the peptide .
the gradient
of the radius of gyration has components on each atom proportional
to its distance from the center of the group , so that atoms close
to the center also experience moderate biasing forces and do not lag
behind the terminal atoms when the biasing force varies rapidly over
time .
a more elaborate discussion of this problem can be found in
ref ( 42 ) . in
some cases
, however , coordinates involving large collection
of atoms will have less resolution than more local ones .
a biophysical
example is permeation through an interface , such as a lipid bilayer .
the common choice of transition coordinate is the distance of the
permeant molecule to the center of the bilayer center , projected onto
the bilayer normal .
in such a case , the physically relevant phenomenon
is interaction of the permeant with the membrane surface , on a local
scale .
if the bilayer patch is large enough , it will experience fluctuations
away from planarity , thus the local position of the interface will
fluctuate with respect to the bilayer center . in turn , this will cause
spurious fluctuations in the transition coordinate . a comparable situation
arose in a study of glycerol permeation through the water channel
protein glpf .
interaction of the permeant
with the protein depended on distances to neighboring pore - lining
residues , which fluctuated with respect to the bulk of the protein .
therefore , the global coordinate measured between the protein and
glycerol molecule had insufficient resolution on a local scale , and
a more local coordinate had to be defined to resolve the structure
of the free - energy profile in the constricted region of the selectivity
filter .
depending on system
size and implementation
details , choices of coordinates may impact performance noticeably .
a common application case is a biomolecular simulation performed with
the namd package , with the adaptive biasing
force algorithm implemented in the collective
variables module .
namd is highly parallelized
and can simulate large systems on supercomputers with nearly linear
scaling .
the current implementation of the adaptive biasing force
algorithm is not parallelized and runs on one node , leading to two
potential bottlenecks : ( 1 ) poor serial performance on the master node :
for the most expensive variables ( e.g. , those involving sums on atom
pairs ) , the bias calculation on the master node might take longer
than the force calculation on other nodes . ( 2 ) scaling may suffer
even for computationally simple coordinates , if too many atom coordinates
and forces have to be communicated across nodes , increasing latency .
this second case may affect any highly parallel application with coordinates
defined on many atoms . in practice ,
one often has to find an acceptable
trade - off between variance and performance by selecting a reasonable
number of cartesian coordinates that are most representative of the
quantity of interest . to describe conformational fluctuations of a
protein ,
for example , the root - mean - square deviation of carbon
coordinates is often a good compromise . to increase
the efficiency of exploring the transition coordinate in adaptive
biasing force or umbrella sampling ,
it is common to break down the transition path
into a series of sequential strata or windows .
this
idea arises from the intuition that the
time to convergence grows as the square of the range of the transition
coordinate .
convergence
of the free energy over the
entire range is achieved
after t0 .
let us now divide the transition
path into n nonoverlapping windows of lengths , for which convergence
is attained after t1 , ... , tn. as shown in the supporting information , t0 >
iti. we illustrate this result
in a simple example of a tagged water
molecule diffusing in a bulk environment over a stretch of 20 .
the transition coordinate is the projection of the distance separating
the centers of mass of the tagged water molecule and the simulation
cell along a given direction of cartesian space .
translational invariance
due to the isotropic nature of the liquid imposes the condition that
the free - energy change along the transition coordinate be zero .
for
this system , the potential of mean force was determined in a single ,
20 long stratum , two 10 strata , four 5 strata ,
and eight 2.5 strata .
the simulations continued until the root - mean - square
deviation between the computed potential of mean force and the reference
zero free - energy profile was less than 0.1 kcal / mol .
as can
be observed in figure 3 , t0 , the time necessary to attain convergence
within this preset tolerance without stratification is on the order
of 100 ns . when the transition coordinate is divided into two strata ,
convergence in each 10 stratum
is reached in approximately
40 ns , i.e. , in 80 ns over the full 20 range .
the effect of
stratification increases further , as the reaction coordinate is decomposed
into four and eight windows . in these cases ,
convergence is achieved
in approximately 6 and 2 ns per window , respectively , which corresponds
to 24 and 16 ns for the complete 20 range .
stratification strategies
for the translationally invariant toy
model of a tagged water molecule diffusing in a bulk aqueous medium .
the transition path spans 20 and is handled in a single window
( a ) , in two 10 windows ( b ) , in four 5 windows ( c ) , and
in eight 2.5 windows ( d ) . for each stratification strategy ,
a potential of mean force calculation is carried out until the root - mean - square
deviation with respect to the accurate zero free - energy profile is
less than 0.1 kcal / mol . both theoretical considerations and
a simple example given
above
appear to suggest that extensive stratification should be always preferred .
first , simulations in
each window require initial equilibration , which may erase benefits
gained from stratification into many windows .
perhaps more importantly ,
extensive stratification may impede ergodic sampling of the phase
space .
this behavior , shared with the standard thermodynamic integration ,
will be discussed in section addressing nonergodicity
scenarios .
in contrast to umbrella sampling and its adaptive
variants , the
adaptive biasing force algorithm does not require that consecutive
windows of a dissected transition coordinate overlap .
provided that
convergence has been achieved in each window , the gradient , a( ) , can be reconstructed merely by joining the gradients
from individual windows at the boundaries .
this improves efficiency ,
as the requirement for overlap between windows may frequently add
as much as 50% to the total simulation time .
if the gradient between
consecutive windows is not continuous to within statistical error ,
this is usually a sign of difficulties with ergodic sampling .
again ,
this problem will be considered in section addressing
nonergodicity scenarios . in this section ,
we
examine the convergence properties of the adaptive biasing force algorithm
and the reliability of the computed free - energy estimates .
first ,
the reader is invited to follow a demonstration that the numerical
scheme formally converges .
then , we discuss how statistical errors
associated with the reconstructed free - energy landscapes can be measured
and managed .
the aim of this section is to explain convergence properties of
the adaptive biasing force algorithm . for more details ,
we
consider the overdamped langevin dynamics,16where xt is defined in the n - dimensional
torus ( namely , in [ 0 , 1 ] with periodic boundary
conditions ) and (x1, ...
we direct the reader to refs ( 67 ) and ( 68 ) for extensions to more
general situations of the results presented below . starting
from eq 16 and using the above choice
of the transition coordinate
, the adaptive biasing force dynamics
can be represented as17where xt1 denotes the
first coordinate of the vector xt , e1 is the vector with coordinates ( 1 , 0 , ... , 0 ) ,
and 1v denotes the partial derivative
of v(x1, ... ,xn ) with respect to x1 . as explained in the section theoretical backdrop ,
it is clear at least formally that the
only possible stationary
state for at is the free
energy ( up to an additive constant ) . indeed ,
if at converges to some stationary state a , then the law of xt converges to z1 exp({v(x )
a[(x ) ] } ) dx , which implies that ( 1v(xt)xt1=x1 ) is a(x1 ) .
yet , this does not provide a proof of the convergence of at to a , and it does not explain
why the adaptive biasing force dynamics of eq 17 indeed converges faster to equilibrium than the original , unbiased
dynamics of eq 16 . one way to understand
for the original dynamics
of eq 16 , the density satisfies the
fokker
planck equation:18for the adaptive biasing force dynamics ,
the
density satisfies19 it ought to be noted that eq 19 is a
nonlinear
partial differential equation ( pde ) , which makes the study of its
long - time behavior much more complicated than for the linear fokker
using appropriate mathematical
tools ( namely , entropy techniques ,
see the supporting information ) , one can
show that if the transition coordinate is well chosen , the convergence
to equilibrium for the adaptive biasing force dynamics of eq 19 is much faster than for the original unbiased dynamics
of eq 18 . roughly speaking
, the assumption on
the transition coordinate is that the canonical measure z exp[v(x ) ] dx is more multimodal
than the conditional measures at a fixed value x1 of the transition coordinate
this
is typically the case for the simple
illustrative two - dimensional potentials in figures 1 and 2 if (x1,x2 ) = x1 .
this can be fully quantified , and it actually gives
a way to measure the quality of the transition coordinate . in
the analysis outlined above
, it is assumed that the conditional
expectation appearing in the adaptive biasing force dynamics is computed
exactly .
this analysis is therefore well adapted to discretizations
that involve many replicas in parallel , which indeed converge to the
adaptive biasing force dynamics with the exact conditional expectation ;
see ref ( 69 ) .
analysis
of the adaptive algorithms ( adaptive biasing force or adaptive biasing
potential ) with estimates of the conditional expectations based on
trajectory averages along a single path are much more complicated .
see refs ( 70 ) and ( 71 ) for preliminary results
for the wang
just like any experimental
measurement , free - energy calculations , of either alchemical or geometrical
nature , ought to be reported with the associated error bars . in the
absence of an error estimate ,
a free - energy difference is generally
of limited utility , making direct comparison with experiment difficult
and speculative .
although much effort has been devoted in recent years
to the characterization of errors that are associated with free - energy
calculations , estimating the reliability of such calculations remains an intricate
task .
this explains why it is not unusual that calculated free energies
are still being published without error estimates .
ideally ,
any free - energy difference should be determined from a series of n independent simulations . if this were , indeed , the case ,
the best possible estimate of the target free - energy difference would
be the expected value over the n simulations , i.e. , , where denotes the estimate of
the exact quantity , a , inferred from one individual
free - energy calculation .
then , the
associated mean - square error can be written as20the first term
of eq 20 , = , is the variance ,
or the precision of the free - energy calculation .
in other words ,
it is a measure of its statistical error . the second term , , is the square
of the bias of the free - energy
estimator , i.e. , the square of the difference between the expected
value of the estimator and the actual free - energy change , a .
the bias , also referred to as the accuracy of the free - energy calculation , is a measure
of systematic error of the latter . how to estimate statistical
error for the adaptive force method
will be discussed in the next section .
one important source
of bias arises from incomplete sampling in finite - length simulations
due to quasi nonergodic behavior of the system .
although this behavior
can not be quantified , in many instances it can be detected .
then ,
a number of remedial tools are at our disposal . how to recognize and
remedy problems with quasi nonergodicity will be discussed in section addressing nonergodicity scenarios .
other common
sources of bias are inaccurate treatment of intermolecular interactions
and algorithmic artifacts arising primarily from imprecise numerical
integration of the equations of motion .
these contributions to bias ,
which are common to all simulation methods , will not be discussed
here .
the adaptive biasing
force method is typically applied to nanoscale molecular processes
under physiological conditions , a realm dominated by thermal noise .
thus , estimating free - energy differences using adaptive biasing force
requires careful consideration of statistical error . within the adaptive biasing force framework ,
free - energy
differences are determined by integrating the estimated mean force
of the system exerted along the transition coordinate .
namely , a free - energy
difference on the interval za zb can be expressed as21where fz is the
average of the instantaneous force on the transition coordinate
at the position (x ) = z. thus , to determine the statistical error of the free - energy differences ,
we must delve into the statistics of the mean system force .
we assume
that the transition coordinate , , is discretized and instantaneous
forces calculated during the course of the simulation are collected
in appropriate bins along . as derived in the supporting information , one way to estimate the error of the
mean force in bin i is given by22where f(xt ) = f(xt )
fi is the random component of the instantaneous force , ni is the number of samples
accrued in bin i , t is the
time step of the simulation , and i and f2i are the autocorrelation time and variance of f(xt ) in
bin i. given a reliable estimate of the error
of the mean force in each
bin , we are prepared to analyze how these estimates are propagated
to yield free - energy differences . on a discrete grid along the transition
coordinate , the integral in eq 21 becomes23where ia and ib are bin indices delimiting the -interval [ za , zb ] .
assuming independent behavior in each bin , the error of
a sum of mean forces is approximated from from the bienaym
formula as equal to the square root of the sum of squares of the errors
of these mean forces,24 a notable property of this formula is that the error increases
with the size of the interval over which the free - energy difference
is calculated . for concreteness ,
we now consider the error in free - energy differences for the reversible
folding of deca - alanine in vacuum .
we define the transition coordinate
of interest as the end - to - end distance of the peptide , specifically
the distance between the carbonyl carbon atoms of the first and the
tenth residue .
below we discuss the behavior of the three quantities
that enter eq 22 , namely .
the number of samples
in each bin ni , the standard
deviation of the random force ( f2i ) , and the autocorrelation
time of this force i .
the black curve in figure 4a is the number of samples in each bin of width = 0.1 for the adaptive biasing force
calculated in the range from 12 to 32 ( the red curve will be
discussed later in the paper ) .
the number of samples , ni , in this range varies from 17 000
to 36 000 .
thus , as expected from the adaptive biasing force
algorithm , the number of samples in each bin approaches uniformity .
in this case ,
nonuniformity of sampling exceeds only slightly a factor
of 2 , which corresponds to the variations of the biased free energy
not exceeding 0.5 kcal / mol . for comparison , the unbiased free energy
changes in the same range by approximately 30 kcal / mol .
in figure 4b , we show the distribution of
instantaneous forces in four different bins .
the forces in each bin
are approximately normally distributed , with similar standard deviations
of about 20 kcal mol .
this point is underscored in figure 4c ,
which illustrates that , for deca - alanine in vacuum with langevin dynamics
emulating buffeting of the molecule by solvent , the standard deviation
of the force acting along the transition coordinate changes very modestly ,
even though the peptide explores structures that are as disparate
as can be imagined .
the peptide courses through different compact
forms for < 10 , remains mostly -helical for
10 < < 16 , and forms extended structures with
diminishing helical fractions as increases beyond 16 .
the approximate uniformity of f2i seen here
is also characteristic of many other systems .
for example , it has
been previously found that the standard
deviation of the instantaneous force on the center of mass of a water
molecule is about 2 kcal mol , irrespective of whether the molecule lies in the bulk aqueous phase
or in the hydrophobic core of a lipid bilayer . on the other hand ,
the transfer of a solute across a liquid vapor interface is
an example of a transition for which f2i is expected to vary considerably
with . coordinate dependence of sampling and the system force
distribution
in reversible folding of deca - alanine .
( a ) samples in each bin for
a 10 ns adaptive biasing force calculation on the domain
[ 4 , 32 ] ( black curve ) and a 100 ns calculation on the domain
[ 4 , 32 ] ( red curve ) . note the logarithmic
scale on the vertical axis .
( b ) distribution of the -component
of the instantaneous system force for different ranges of .
( c ) standard deviation of the -component of the instantaneous
system force as a function of . note that f2i is considerable .
because this term enters prominently
the expression for statistical error in eq 22 , there are significant merits in reducing the dispersion of instantaneous
forces .
first , because the expression for the ensemble average of instantaneous
force is not unique we can , in principle , choose one that reduces
f2i .
second , variation of forces can be also reduced by a thoughtful
choice of the transition coordinate .
the third variable in eq 22 , the correlation
time of the instantaneous system force i , is also the most difficult to calculate .
the sampling in
a single bin is rarely sufficient to obtain a converged autocorrelation
function of the system force ; thus , in figure 5a we plot the autocorrelation function averaged over 40 bins along
different regions of . the correlation time for each region ,
, is determined by fitting an unscaled exponential function
e to the positive values
of the autocorrelation function .
coordinate
dependence of correlations in the system force in reversible
folding of deca - alanine .
( a ) autocorrelation functions of the random
component of the instantaneous system force for different ranges of
. the functions are normalized by the variance f2i
( b ) correlation time for -ranges
in panel a. using the calculated
values of ni , f2i and i , we now estimate the uncertainties
of free - energy differences between deca - alanine structures with different
end - to - end distances .
the mean system force , with uncertainties calculated
by eq 22 , is shown in figure 6a .
because only free - energy differences between two points
along , rather than free energies at single points , have a
clear physical meaning , we focus on errors associated with these differences .
to compute the error in the free - energy difference at points za and zb
, we must accumulate the uncertainties of
the force between these two points , in accord with eq 24 .
if stratification is used and points za and zb are in different windows , then the bienaym formula
needs to be used across all windows separating these points .
propagation
of error in the mean force to the error of free - energy
differences .
( a ) mean system force on for a 10 ns adaptive
biasing force calculation , with error bars determined according to
eq 22 .
we show the estimated
error
of a between the -helical minimum free - energy
state ( za = 14 ) and other
states within the interval zb [ 12 , 32 ] .
if we want to calculate , for instance ,
the difference in free - energy between the minimum and = 16
, we obtain 3.6 1.8 kcal / mol .
larger distances in
yield larger error : the free - energy difference between the minimum
and the plateau at 25.5 is 19 4 kcal / mol .
it is
expected that , for sufficiently long total simulation time , t , statistical errors of both the average forces and free - energy
differences will decay proportionally as t. the same dependence on t should apply to deviations from nonuniform sampling .
if force statistics
is collected in m bins , then the root - mean - square
deviation from the uniform distribution can be defined as the square
root of the variance , var(t),25where nstepk and nstep are the number of samples
in bin k and the total number of samples at time t , respectively . for large t , the variance
is expected
to be proportional to 1/t or , equivalently to 1/nstep . in other words ,
an
example of how var(t ) behaves in a typical simulation
is shown in figure 7 .
errors that clearly deviate from such
behavior are a sign of insufficient sampling . if the problem persists
with increasing t , and especially if errors exhibit
large fluctuations , then most likely problems with quasi nonergodic
behavior have been encountered .
var(t ) as a function of the number of molecular dynamics
steps , nstep .
this quantity was calculated
for the permeation
of k through a transmembrane hexametric channel of a peptaibol ,
trichotoxin in a window along the normal to the membrane spanning
the range between z equal to 15 and 9
.
note that for nstep between 0.6 10 and 2.3 10 , nstep var(t ) differs
from its average value in this range by no more than 10% .
the inset :
the number of force samples along z in the window
as a function of nstep .
a common manifestation of pathological free - energy calculations ,
in particular those of geometrical nature , is quasi nonergodicity ,
wherein sampling along the selected transition coordinate appears
to be hampered . here
, we inspect closely the effects that impede accurate
results to be obtained from free - energy methods , including the adaptive
biasing force scheme ( notably hidden barriers in the slow manifolds ) ,
and discuss how to identify these effects and outline possible remedies ,
by increasing the dimensionality of the transition coordinate , improved
stratification , or sampling aided by multiple - replica strategies .
the primary objective of importance - sampling schemes is to facilitate
exploration of the transition pathway with a uniform probability . among these schemes ,
as has been previously emphasized , the adaptive
biasing force algorithm uses a local estimate of the gradient , a , acting along the transition coordinate , to erase
progressively the original ruggedness of the free - energy landscape .
as has already been discussed in section formal
convergence of the adaptive biasing force algorithm , this feature
is valid from a theoretical standpoint .
how true is this in practice ?
in most instances , satisfactorily uniform sampling is achieved quite
efficiently . occasionally , however , the adaptive biasing force algorithm
does not perform as expected .
the reminder of this section is devoted
to explaining and identifying these special , yet important cases .
potential difficulties in applying the adaptive biasing force algorithm
are intimately related to the choice of transition coordinate .
a basic ,
yet seldom verified assumption that underlies this choice is the separation
between time scales of motions along the transition coordinate and
orthogonal degrees of freedom ( see section transition
coordinates and rare events ) .
for complex , rugged free - energy
landscapes , notably those formed by parallel valleys separated by
considerable barriers in the direction orthogonal to the transition
coordinate ( figure 8) , assuming time scale separation may turn out to be unwarranted . because
the adaptive biasing force algorithm exerts no direct action in the
orthogonal space , it will not improve sampling at constant .
returning to the foundational expression for the adaptive biasing
force method , eq 8 , which relates the gradient
of the free energy to an ensemble average at constant value of the
transition coordinate , the inability to cross hidden barriers in the
orthogonal space is tantamount to incomplete ensemble averages and ,
hence , poor estimates of free - energy changes .
common free - energy landscape
featuring parallel valleys , collinear
to the transition coordinate , . these valleys are separated
by substantial free - energy barriers in the direction , orthogonal
to . can be interpreted as a slow degree of freedom
coupled to and hampering progression along the latter direction .
excessively stratified reaction pathways preclude spontaneous crossing
of high barriers , typically a1 .
wider windows should allow diffusion toward values of , where
the barrier separating valleys in the direction is smaller ,
typically a2 .
their presence is a guaranteed
sign of flawed free - energy calculations , but their absence is not
a sufficient condition to ensure that such calculations converged
to the correct value . as has been discussed previously , sampling along
the transition coordinate in well - behaved simulations should approach
uniformity with time , and the statistical error associated with the
biasing force or , equivalently , the free - energy differences should
decrease in a predictable fashion . if this is not the case , difficulties
in equilibrating the system along orthogonal degrees of freedom are ,
most likely , at play . to illustrate this point ,
we return to the toy
model of deca - alanine reversibly folding in vacuum and to figure 4a . in the range of between 12 and 32 ,
in contrast ,
in the [ 0 , 12 ] range , sampling remains quite nonuniform , even
after 100 ns ( red curve ) .
as has already been pointed out in the section transition coordinates and rare events , there
are a number of metastable states in this region , all corresponding
to similar values of . difficulties in properly averaging over
these states markedly impedes equilibration along . in the
context of stratified simulations , the presence of hidden barriers
along degrees of freedom orthogonal to often leads to discontinuous
biasing forces between adjacent windows .
another strategy for
exposing apparent nonergodic behavior is to
carry out bidirectional calculations , i.e. , initiate the adaptive
biasing force simulation from both end points along the transition
coordinate . just as in free - energy perturbation calculations ,
the resulting hysteresis is a good indicator
( although not necessarily a measure ) of error .
if the hysteresis markedly
exceeds statistical error , the calculated free - energy values are ,
most likely , poorly converged .
in such a case , simply combining data
from both directions is not likely to improve accuracy significantly ,
as their proper weighting remains unknown .
when quasi nonergodicity
scenarios are encountered , additional
simulation strategies , such as multiple walkers or multidimensional
transition coordinates , should be brought to bear .
formally , the adaptive biasing
force algorithm does not prescribe
a particular window size nor does it require that consecutive windows
along a stratified transition coordinate overlap .
provided that convergence
has been achieved in each window , the gradient , a( ) , can be reconstructed merely by joining the gradients from
individual windows at the boundaries .
as we have already argued , from
the efficiency point of view it might appear that using small windows
is always beneficial .
one
concern about extensive stratification is that efficient sampling
of rugged landscape along orthogonal degrees of freedom may require
temporary excursions beyond the window s boundaries , where
the barrier separating adjacent valleys is smaller .
the barrier between the minima
along the orthogonal degree of freedom , , may be difficult
to cross at in the range of [ 0 , 0.2 ] , but not in the [ 0.8 ,
1 ] range . in this case , stratification will create kinetic traps ,
most likely reducing rather than improving efficiency .
a similar
problem was observed in simulations aimed at determining the potential
of mean force for a small , proline - rich peptide , p41 , bound to the
sh3 domain of abl kinase using the root - mean - square deviation ( rmsd )
with respect to the native conformation as the transition coordinate .
there
, a second minimum was only discovered through creation of a
window that did not encompass the native state ( see figure 2a in ref ( 89 ) ) .
this second minimum
represents a shift in register of the peptide in its binding site ,
and although it would occur spontaneously given sufficiently long
time , its probability can be enhanced by first driving the peptide
to higher values of rmsd , which disrupts some of the bonds characteristic
of the native state , and then letting it come back . because
it is often not possible to predict orthogonal barriers a
priori , determining an appropriate windowing scheme typically
requires an adaptive procedure .
the basic criterion here is the continuity
of the biasing force across consecutive windows .
if one is concerned
about quasi nonergodicity , a good strategy is to start with large
windows . if the continuity of forces appears to be satisfactory , one
might attempt to improve efficiency through further stratification .
the advantage of this strategy is that the approximation to the biasing
force acquired from the large - window simulation can be used in smaller
windows .
if no windowing scheme yields continuous forces , then other
strategies , described in the next section , should be employed .
simulations involving multiple
replicas are perhaps the most powerful strategies to accelerate ergodic
sampling along degrees of freedom orthogonal to the transition coordinate .
beyond the embarrassingly parallel strategy of running
independent simulations to obtain more sampling than is possible with
a single simulation in the same real time , a number of schemes have
been devised that significantly enhance sampling , at the cost of transferring
information between replicas . in a prototype example of multiple - replica
algorithms , originating with monte carlo simulations , replicas are run at different temperatures and exchanges of system
configurations are attempted periodically between replicas , so as
to maintain a canonical ensemble at each temperature .
the advantage of this method is that replicas at higher
temperatures cross energetic barriers more quickly and can pass the
resulting configurations to replicas at lower temperatures , preventing
the latter from remaining in metastable states .
exchange of replicas
with different umbrella - sampling potentials , known as hamiltonian
exchange , has also been widely successful . with
the adaptive biasing force method , each replica
multiple - walker strategies range from simply running similar independent
adaptive biasing force calculations in parallel , to more complex ones ,
involving communication between replicas . here
we consider two communication strategies that can be used in
concert . in the first strategy , which we refer to
as shared adaptive
biasing force , the instantaneous forces sampled from each walker are
collected in a single shared buffer as the simulations progress simultaneously .
in the current implementation ,
the shared buffer is merely conceptual :
each walker retains its own buffer , which is synchronized with all
the others at fixed intervals .
regardless , shared adaptive biasing
force can result in significantly faster exploration of the transition
coordinate and improved convergence of the free energy , as compared
to the case for independent walkers .
a second strategy ,
complementary to the first , is the application of so - called walker
selection rules .
these selection rules eliminate replicas with values
of the transition coordinate that are already well sampled , while
duplicating replicas in relatively unexplored regions , enforcing more
uniform sampling .
selection rules may be implemented using a
so - called resampling
procedure and weights that are associated with each replica .
there are many ways to choose the weights and
to implement this idea in practice .
the basic requirement is that
the selection mechanism automatically vanishes at equilibrium , namely
when the biasing force is the mean force .
here , we calculate the weight
of each walker in a somewhat simpler way than in previous studies , while obtaining similar results .
the weight assigned to each walker
is the inverse of the number of samples accrued in a neighborhood
of bins near the walker .
specifically,26where j is the bin occupied
by walker i at the time of application of the selection
rules , nj(t )
nj(tlast ) is the number of samples that have been
accrued in bin j since the last selection and resampling
step , and h defines the number of bins surrounding j that are included in the sum .
the walkers are resampled
on the basis of these weights , and the selection mechanism is switched
off when the smallest and largest ni(t ) differ by less than 20% , i.e. , when [ max(1/wti )
we consider
reversible unfolding of deca - alanine as a model system for testing
different multiple - walker strategies . in figure 9a
we compare the results of adaptive biasing force calculations of
8 ns total simulated time to a converged free - energy profile .
a single ,
8-ns long simulation gave the poorest results , whereas 16 short , independent
calculations perform somewhat better , even though the region 31.432
has not been sampled at all . in contrast , shared adaptive biasing
force yields complete and more uniform sampling , and a smoother , more
reliable free - energy profile .
the best free - energy profile , almost
indistinguishable from the converged one , was obtained from shared
adaptive biasing force combined with walker selection .
note that ,
as expected , the free energies converge to the same values for sufficiently
long simulations , irrespective of the multiple - walker strategy , as
demonstrated in figure 9b .
an implementation
of the shared adaptive biasing force algorithm with the selection
rules used here is expected to be available in future releases of
the molecular dynamics program namd .
( a ) potentials of mean force obtained for different multiple - walker
strategies and total simulation times of 8 ns . for reference ,
the
black curve shows the result for a total simulation time of 128 ns .
single refers to a single long 8 ns simulation , and
independent denotes the result of combining force
samples for 16 walkers after they ran independently for 0.5 ns . for
the curves marked shared and selection ,
force samples were synchronized among walkers every 20 ps .
also included the application of walker selection , as described in
the text , on the same interval .
( b ) potentials of mean force obtained
for different multiple - walker strategies and total simulation times
of 128 ns .
there are several
reasons to perform
adaptive biasing force calculations with a transition coordinate of
dimension greater than one .
first , we may be simply interested in
how the free energy varies as a function of more than one coordinate .
for example , to study the interaction between two molecules , it may
be of interest to obtain the free energy as a function of both their
distance and relative orientation .
as described in section hidden barriers and other challenges to obtaining accurate results , sampling along a single collective variable may be hampered by barriers
in orthogonal dimensions .
if one can identify these orthogonal dimensions ,
the application of adaptive biasing force along them will remove the
barriers and improve sampling . by itself , obtaining a multidimensional
free - energy landscape requires more sampling than is needed to calculate
a one - dimensional profile .
for example , if one uses n bins along collective variable and m bins
along collective variable , each component of the gradient
over both collective variables , a( ,
) , requires mn bins . with one sample
obtained
per time step , the time required to generate a statistically significant
number of samples in all bins will increase approximately proportionally .
despite the additional , substantial computational effort , adding a
second or third dimension may improve efficiency , as it accelerates
convergence and increases uniformity in sampling by allowing the system
to more rapidly cross between multiple parallel valleys
subsequently , one can recover the potential
of mean force along a single dimension through integrating out the
additional dimensions .
however , when
one wishes to include a large number of dimensions , the generalized
adaptive biasing force algorithm ( see section the
generalized adaptive biasing force algorithm ) may be more appropriate .
if the transition coordinate involves angular degrees of freedom
that span their full range ( e.g. , [ 0 , 2 ] ) , an additional complication
develops .
the exact forces and free energies are periodic in these
variables , but this is not necessarily true for these quantities burdened
with statistical errors . one way to restore the required periodicity
is to approximate the free energy as a function of angular variables
with spline functions , coefficients of which are computed to distribute
errors smoothly across the whole hyperspace spanned by these variables .
how to do this has been described by darve et al . as an example
application of multidimensional adaptive biasing force , we consider
the free - energy landscape of deca - alanine as a function of the root - mean - square
displacement ( rmsd ) from three reference structures .
figure 10a shows these reference structures : an -helix ,
a 310-helix , and a particular compact conformation that
we refer to as the conformation due to its visual similarity
to this greek letter . in agreement with the one - dimensional calculations ,
extensively discussed above , the free - energy minimum occurs when the
structure is close to the -helix , i.e. , when rmsd = 0.3 . in figure 10b the three - dimensional
potential of mean force
the violet region on the left
corresponds to the -helix , whereas the region on the right
is closer to the conformation than to the other two structures .
the minimum in the latter region is only 1.2 kcal / mol higher than
the minimum for -helix and lies at ( rmsd rmsd310 rmsd ) = ( 4.3 5.0 2.7 ) . in figure 10c ,
the multidimensional free - energy
landscape thus yields more insight into the correspondence between
the free energy and molecular conformations , revealing , in particular ,
a minimum for a compact structure that was not identified when the
one - dimensional transition coordinate was used .
the root - mean - square deviation
of selected atoms from their positions in each of the three reference
structures defines each of the three transition coordinates .
( b ) free - energy
isosurfaces as a function of the three transition coordinates .
the
violet , pink , and gray surfaces contain all points for which the free
energy is less than 5 , 10 , and 20 kcal / mol , respectively . in all cases
the tick marks represent a root - mean - square deviation of 1 .
the minimum value of the free energy , which occurs at ( rmsd rmsd310 rmsd ) = ( 0.3 2.6 2.7 ) ,
is defined to be zero .
( c ) another view of the surface shown in panel
b , with representative structures shown for the two energetically
favorable regions .
previously ,
we considered one particular approach to multidimensional adaptive
biasing force . here
first , it
is easy to check that if the dimension of the transition coordinate
is larger than one , the biasing force can not be in general derived
from a gradient ( it is not conservative ) . on the other hand , this is of course true of the expected long - time
limit . a natural idea is , therefore , to project , using , for example ,
the classical helmholtz
hodge projection : change the biasing
force ft to t where t = arg min ft l .
because this is consistent with the expected stationary
state , this should not alter the convergence properties .
moreover ,
the interest of this projection is that the variance of the force
is reduced , because the nonconservative part is set to zero .
second , it would be interesting to develop efficient
adaptive biasing force techniques for higher dimensional transition
coordinates .
the standard implementation of the adaptive biasing force
algorithm is currently limited to a dimension up to 3 , because it
relies on a cartesian grid of the transition coordinate values , whose
complexity is exponential with respect to the dimension of the transition
coordinate .
it would not be very useful , however , to develop a technique
that yields a flat energy landscape along the transition coordinate
if the dimension is high , as mapping a high - dimensional cube would
be quite time - consuming .
one alternative idea , explored in ref ( 101 ) , is to use a bias of
the form i=1matii , where
( 1 , ... , m ) denotes
an m - dimensional transition coordinate . under appropriate
assumptions
, one can show the convergence of the method , and preliminary
numerical results are encouraging ; see ref ( 101 ) .
another route would be to consider a bias
of the form i=1matii , or
even following greedy algorithms and tensor product approaches used
in nonlinear approximation theory ( see , for instance , ref ( 102 ) ) , k1i=1mati , ki , where the functions ( a , ... , a)k1 would be
iteratively computed .
the objective of this section is to assess the
influence of geometrical
restraints on the free - energy landscape and how such restraints ought
to be treated in the context of adaptive biasing force simulations .
toward this end
, the nature of the forces at play , either thermodynamic
forces or forces arising from external harmonic potentials , will be
clarified , and the current strategy for handling the latter , using
an extended - lagrangian formalism , will be outlined .
one subtlety of the adaptive
biasing force algorithm that is often
overlooked is its dependence on the measure of the thermodynamic force ,
which is not necessarily synonymous with the total force acting on
the transition coordinate . for most biomolecular simulations ,
for example , hydrogen - bond lengths are often constrained in a simulation
via the rattle algorithm .
if only the
heavy atom of the bonded pair is involved in the transition coordinate ,
then the adaptive biasing force algorithm will include the constraint
force emanating from the hydrogen in addition to the thermodynamic
force , thus contaminating its estimate .
a straightforward solution
is to include both the hydrogen and its parent atom in the collective
variable(s ) defining the transition coordinate , causing the constraint
forces to cancel each other . in other words ,
if the constraint / restraint
force is zero in the collective variable s center of mass ,
it will not contribute to the measured potential of mean force .
alternatively ,
if restraints nonuniformly affect atoms in the collective variable(s )
but are not accounted for in the thermodynamic force used by the adaptive
biasing force algorithm , then convergence is impossible ; the adaptive
biasing force scheme can not remove forces that it does not measure . though it may seem apparent that one will always want to calculate
what the potential of mean force would be in the absence of artificial
restraints , there are cases in which externally imposed restraints
are meant to be included .
ligand
binding free energies , which utilizes a staged procedure involving
a series of geometrical restraints . by design
of the procedure
, these restraints must involve the same atoms also
being biased at each stage and their contributions are individually
determined and tabulated at the end . in this case , to ensure that
all necessary forces are included , a procedure such as extended adaptive
biasing force is required . as mentioned
above , it may be cumbersome to write an analytical expression for
the instantaneous force in dimensions larger than one or for complicated
transition coordinates .
one possible way around this issue is to extend
configurational space with a fictitious degree of freedom
and define an extended potential,27where k > 0 is a ( large )
force constant that couples to the transition coordinate ,
and proceed utilizing the extended transition coordinate , (x, ) = , instead of .
it can be checked that the free energy associated with this extended
system is the convolution of the original free energy with a gaussian
kernel , a( ) = dz k(z ) a(z ) , where
k(z ) = z exp[(/2k)|z| ] is the gaussian kernel
with variance k. the constant k should thus be chosen sufficiently large to ensure that a is a good approximation of a. this enables quick convergence to equilibrium .
the adaptive
biasing force algorithm can be applied to the extended system , the potential energy being v and the transition coordinate being . notice that the instantaneous force on the extended degree of freedom ,
corresponding to free energy a , is
trivial to compute , because it is equal to the harmonic spring force .
the original free energy a can then be recovered
by using either deconvolution procedures or simple unbiasing techniques based on formulas such as eq 34 .
provided that a good estimate of the biased
marginal of the transition
coordinate , (z ) , has been collected ,
the following unbiased estimator of the free - energy derivative can
be used,28where z is the conditional average value of for
(x ) = z. here , the biased marginal
of is used as a correction to the inaccurate biasing force .
it should be noted that this is valid in a more general context , including
the absence of biases ( removing the second term of the right - hand
side ) , in which case one recovers the trivial histogram - based estimator
of the potential of mean force .
although the adaptive biasing force algorithm
is often considered
merely a free - energy calculation technique , it is , at its core , an
importance - sampling scheme that can have applications beyond obtaining
free energies .
one such application is to calculate kinetic parameters ,
such as the diffusivity along a transition coordinate , where an importance - sampling
scheme is often essential to obtain reliable estimates in all regions
of the coordinate , particularly near all - important free - energy barriers .
together , the diffusivity and free energy as functions of the transition
coordinate can be used to construct a kinetic model of a process of
interest , which yields insight beyond the static picture of molecular
phenomena given by free - energy calculations . in this section ,
we describe
a recently developed scheme leveraging adaptive biasing force to compute
simultaneously the free energy and diffusivity along a transition
coordinate .
this scheme is just an example , as a number of other approaches
in which the adaptive biasing force is used to improve kinetic descriptions
of different systems are being investigated .
also note that , although
this scheme was designed with the adaptive biasing force method in
mind , it can be straightforwardly applied to other importance - sampling
techniques . determining kinetic parameters can be more subtle
than mapping
the free - energy landscape , because kinetic descriptions are usually
approximations and can not be exactly derived from statistical mechanics .
a commonly invoked diffusive model is overdamped
langevin dynamics , in which the following equation of motion is assumed for a set of m collective variables z = ( z , z , ...
, z),29where ti is the time derivative
of the ith collective variable at time t , dij(zt ) is the ( i , j ) component
of the diffusivity tensor for the configuration of the collective
variables at time t , fj(zt , t ) is the total force on collective variable j for the configuration at time t , j = /z , and i(t ) is a stochastic variable with i(t ) j(t) = 2dij(zt ) (tt ) .
the total force on variable j , fj(zt , t ) , is the sum of the biasing force , fjbias(t ) , and the system force ,
which can be expressed as the negative of the gradient of the potential
of mean force fj(z ) = ja(z ) . here
, we will focus on diffusive
models along a single collective
variable , but note that multidimensional transitions have been analyzed
by similar approaches . in one dimension
, the overdamped langevin model has two free parameters ,
the free - energy profile a(z ) and
the position - dependent diffusivity d(z ) .
the adaptive biasing force method , as well as other techniques ,
provides a route to a(z ) , whereas
a number of methods have been applied to determining position - dependent
diffusivity .
however , some of the most basic methods are not compatible with
nonuniform free - energy landscapes , and
others are based on the assumption that the free energy can be approximated
as a harmonic well . in methods specifically designed for compatibility with the adaptive
biasing force method , it is assumed that the dependence of diffusivity
on position is weak . to parametrize a
diffusive model , a(z ) and d(z )
should be determined consistently ,
because , for example , coarsening a(z ) results in a reduction of the associated d(z ) .
furthermore , to save computational
resources , it would be desirable to obtain both the free energy and
the diffusivity in the same calculation
. however , in many methods
cited above , equilibrium or steady - state statistics is assumed , which
is likely to yield erroneous results with time - dependent biasing forces .
solutions to the problem of calculating diffusivity in simulations
with time - dependent biases take advantage of statistical tools such
as maximum likelihood or bayesian inference .
conceptually , these methods rely on optimizing
the parameters of the diffusive model such that the observed trajectory
has the greatest likelihood of occurring .
the bayesian inference
scheme described below begins with assuming a particular dynamical model for the collective
variable ( or collective variables ) of interest . in practice , we represent
the functions dij(z ) and fi(z ) by piecewise cubic interpolation from a discrete grid in m - dimensional z - space .
we seek the optimal parameters comprising the values of the
functions dij(z ) and fi(z ) at each grid node that best correspond to the simulated trajectory ,
denoted by t. for simplicity , we represent these optimal
parameters as h*. we consider the trajectory as a set
of discrete hops of duration t . given trial
parameters h , as well as the biasing forces f(t ) , we compute for each hop p ( { z+1 , t+1}{z , t } , f(t ) , h0 ) , the conditional
probability of arriving at transition - coordinate configuration z+1 at time t+1 , given that the system occupied the configuration z at time t.
the probability of the complete trajectory given the parameters is
the product of the probabilities at each step30this equation yields the
probability of the
trajectory given an assumed set of parameters . using bayes
theorem
, we can infer the probability
of the parameters given the trajectory:31 an
advantage of the bayesian approach
is that it permits inclusion of any prior knowledge about the form
of the parameters in a consistent way by defining the prior probability pprior(h ) of the parameters .
for example , one can assume scale invariance of the function values or smoothness of the functions .
finding the optimal parameters
then becomes a problem of finding the set of parameters h * that maximizes posterior probability p(h*|t ) , which can be carried out by generating a markov
chain of states hk using
the metropolis hastings algorithm . in the one - dimensional
case ( or for independent motions along multiple dimensions ) , we can
simplify and discretize eq 29 to yield32where gt is a random
variable with a standard normal distribution .
from the properties of gt
, it is evident that eq 30 becomes33below ,
using this formula and eq 31 , we are able to
reconstruct d(z ) and f(z ) for deca - alanine ,
yielding a complete diffusive model .
indeed , we may even set f = 0 and
construct the diffusive model from an equilibrium simulation .
however ,
the accuracy of the results of a bayesian scheme are wholly dependent
on the quality of sampling near each point along z , necessitating importance sampling for rugged free - energy landscapes .
in the supporting information
, we discuss
assessment of the precision and reliability of the results of the
bayesian scheme , which includes estimating the statistical error ,
as well as checking the consistency of the diffusive model with itself
and the output of adaptive biasing force .
the z - dependent diffusivity
for reversible unfolding of deca - alanine
is shown in figure 11b .
the variation with z could not have been inferred in any simple way from knowledge
of the system force or free - energy profiles .
diffusion along z , which can be thought of as the rate at which the end - to - end
distance randomly changes , is highest near z = 16
, corrsponding to slightly unfolded -helical structures .
the ensemble of diverse compact structures in the range z [ 5 , 10 ] seems to be associated with the lowest diffusivity
values , whereas a secondary minimum appears near z = 22 .
given these data , one might hypothesize that the diffusivity
is inversely related to the conformational degeneracy at z. note that figure 11b shows considerable
dependence of the calculated diffusivity on t , the time over which the trajectory is discretized in eq 33 , which implies that the motion on the observed
times is not well modeled by overdamped langevin dynamics , and that
a more sophisticated model of motion along z may
be needed .
( a ) comparison of the system force as determined
by adaptive biasing force and that calculated from the bayesian scheme
on the same trajectory .
( b ) diffusivity as a function of the end - to - end
distance of deca - alanine for different observation time intervals
t .
free - energy calculations have become
standard tools of statistical
mechanics applied to a wide variety of problems in chemistry and biology .
this has been possible due to significant theoretical advances in
this area , their highly efficient implementations in software packages
developed for modern , parallel computers , and remarkable improvements
in computational power available for free - energy calculations . yet
,
these calculations are still not sufficiently mature to be carried
out without careful supervision of the end user .
this may be fortunate ,
as there is no substitute for physical insight of the researcher .
this also implies that a number of good practices should be followed in free - energy calculations in general , and in
applying the adaptive biasing force in particular .
these good practices
are usually quite simple and involve careful design of simulations ,
monitoring their progress and postprocessing analysis . in favorable
circumstances , they will simply increase confidence that the calculated
free energies are reliable . in less favorable cases , they are even
more important , as they allow for identifying and correcting shortcomings
of the calculations that might adversely affect the results and their
interpretation .
below , we recapitulate these good practices , which
have been already discussed in a considerable detail in the preceding
sections.(1)careful
choice of the transition coordinate
is a key step toward successful free - energy calculations . in making
this choice , it is desirable not only to capture the physical nature
of a problem of interest but also to ensure small variance and smoothness
of the biasing force .
ignoring the latter
issues may adversely affect efficiency and even correctness of the
calculation . reducing the variance
should be balanced with the cost
of calculating the instantaneous value of the transition coordinate ,
as usually the former will decrease and the latter will increase with
the number of atoms involved .
this balance will depend on implementation
details and , therefore , some familiarity with the underlying code
might be required.(2)a suitable stratification strategy
should be planned in advance .
if there are concerns about possible
quasi nonergodic behavior of the system , and in particular the existence
of parallel channels between the end point states , it is recommended
to use large windows .
if these concerns prove to be unjustified , it
is always possible to switch to smaller windows without losing information
about the biasing force that has already been accrued . in other instances
it is usually more efficient to stratify the transition coordinate
into smaller windows throughout a simulation.(3)free - energy calculations should always
be accompanied by estimates of statistical errors . for the adaptive
biasing force algorithm , a formula to do so exists and should be applied
whenever possible . without error estimates the reliability of the
calculated free - energy values is questionable and the ability to compare
them with experimental measurements is seriously hampered.(4)in stratified simulations ,
lack of
continuity in the biasing force across consecutive windows that clearly
exceeds statistical errors is a sure sign of problematic free - energy
calculations .
this issue should not be ignored , and any attempt to
circumvent it by way of some sort of averaging is unlikely to succeed .
instead , remedial steps aimed at improving ergodic behavior of the
system , such as applying the multiple - walker strategy , should be taken.(5)it is usually very useful
to monitor
the behavior of the total , biased force , as it should converge to
zero in long simulations . moreover , the rate of convergence at sufficiently
long times t should become proportional to 1/t . if the biased force along the transition coordinate or
within a window exhibits large deviations from nonuniformity , which
clearly does not decrease with time , or the convergence rate is erratic ,
we have , again , a likely indication of quasi nonergodicity in orthogonal
degrees of freedom .
then , there is no basis for assuming that a reliable
free - energy dependence on the transition coordinate can be extracted
from the calculated forces . instead , in such circumstances , techniques
for removing quasi nonergodicities along orthogonal degrees of freedom
should be brought to bear .
careful
choice of the transition coordinate
is a key step toward successful free - energy calculations . in making
this choice
, it is desirable not only to capture the physical nature
of a problem of interest but also to ensure small variance and smoothness
of the biasing force .
ignoring the latter
issues may adversely affect efficiency and even correctness of the
calculation . reducing the variance
should be balanced with the cost
of calculating the instantaneous value of the transition coordinate ,
as usually the former will decrease and the latter will increase with
the number of atoms involved .
this balance will depend on implementation
details and , therefore , some familiarity with the underlying code
might be required .
if there are concerns about possible
quasi nonergodic behavior of the system , and in particular the existence
of parallel channels between the end point states , it is recommended
to use large windows .
if these concerns prove to be unjustified , it
is always possible to switch to smaller windows without losing information
about the biasing force that has already been accrued . in other instances
it is usually more efficient to stratify the transition coordinate
into smaller windows throughout a simulation .
free - energy calculations should always
be accompanied by estimates of statistical errors . for the adaptive
biasing force algorithm ,
a formula to do so exists and should be applied
whenever possible . without error
estimates the reliability of the
calculated free - energy values is questionable and the ability to compare
them with experimental measurements is seriously hampered . in stratified simulations ,
lack of
continuity in the biasing force across consecutive windows that clearly
exceeds statistical errors is a sure sign of problematic free - energy
calculations .
this issue should not be ignored , and any attempt to
circumvent it by way of some sort of averaging is unlikely to succeed .
instead , remedial steps aimed at improving ergodic behavior of the
system , such as applying the multiple - walker strategy , should be taken .
it is usually very useful
to monitor
the behavior of the total , biased force , as it should converge to
zero in long simulations .
moreover , the rate of convergence at sufficiently
long times t should become proportional to 1/t . if the biased force along the transition coordinate or
within a window exhibits large deviations from nonuniformity , which
clearly does not decrease with time , or the convergence rate is erratic ,
we have , again , a likely indication of quasi nonergodicity in orthogonal
degrees of freedom .
then , there is no basis for assuming that a reliable
free - energy dependence on the transition coordinate can be extracted
from the calculated forces . instead , in such circumstances , techniques
for removing quasi nonergodicities along orthogonal degrees of freedom
should be brought to bear . following
these simple good practices guarantees the improved quality
of free - energy calculations carried out by way of the adaptive biasing
force method but
does not ensure that all problems encountered in
such calculations , especially those related to quasi nonergodicity ,
have been identified . to this end ,
the toughest challenges to practitioners
of the adaptive biasing force algorithm and related methods are linked
to multiple slow degrees of freedom leading to orthogonal barriers .
one way to address these problems is through a multiple - walker strategy ,
which has proven effective in a test system that exhibits metastability
in the orthogonal space , a significant challenge to the classic adaptive
force method .
along the same lines , the
basic method can be integrated with other enhanced sampling techniques
that involve multiple - copy schemes , such as parallel tempering and
hamiltonian exchange .
another promising
research direction is to increase the dimensionality of the bias ,
as is done in an implicit way in the generalized adaptive biasing
force scheme .
real - life
applications would , however , require an accessible and well - documented
implementation , now available as part of the independent collective
variables module . although better
sampling of orthogonal degrees of freedom is the
most promising direction for increasing the efficiency of the adapting
biasing force method , there is also room for improvement in achieving
convergence along the transition coordinate .
for example , it would
be of interest to develop adaptive algorithms for the early stages
of simulations that would converge faster than the currently used
step or ramp functions .
one possibility along these lines is to employ
a kernel function along . in other words , adaptation in a given
bin would depend not only on samples accrued in this bin but also
on samples in neighboring bins .
another avenue to improve convergence along the transition
coordinate , which has not been explored so far , is to exploit the
freedom in defining the ensemble average of instantaneous force and
identify choices that reduce the variance .
for example , the error in integrating force to calculate free energy
that is due to binning of force values could be reduced by using improved ,
smooth interpolation schemes .
further , we observe that the best accuracy
is obtained not when the free energy is flat but rather when the statistical
error of the average force is the same everywhere along the transition
coordinate . to achieve this , the number of samples , nk , in bin k should be
such that k/(nk ) rather than nk is constant for all bins ,
where k is the standard deviation
of estimated average force in bin k. this is realized
if the biasing potential due to the average force used in the standard
adaptive biasing force method is supplemented by the term 2kbt ln(k/0 ) , where 0 is the standard
deviation at a reference point .
in practice , k and 0 are estimated from their running values
or their approximations during the course of a simulation .
the additional
term becomes important whenever the friction coefficient changes markedly
with or parts of the system undergo large fluctuation , for
example because they are near phase transition .
a number of
extensions to the adaptive biasing force method have
not been explored yet .
for example , the method could be straightforwardly
extended to transformations along a parameter of the hamiltonian if
an equation of motion was associated with this parameter , as is done
in metadynamics .
it would be of interest to check whether such application
of the adaptive biasing force method were more efficient and reliable
than other , related methods currently used for this purpose , such
as conventional thermodynamic integration , metadynamics , or the wang
another extension of the method that was outlined in one
of the original studies on the adaptive biasing force but has not been pursued so far , is to add
to the biasing force another contribution that would depend only on
and would favor certain states of particular interest to the
user or drive the dynamics in a specified direction . the latter would
make the adaptive biasing force method a more efficient and better
controlled alternative to steered dynamics . finally , we emphasize
that the adaptive biasing force is not just
a free - energy calculation technique but can also be seen as a general
adaptive biasing scheme ( see , for instance , ref ( 124 ) for applications in bayesian
statistics ) .
indeed , once a correct sampling of the biased measure zp1 exp(pmp/2 ) dp zt01 exp{[v
at0](x ) } dx has been obtained ( t0 being a fixed time , and the bias being fixed from time t0 : t t0 , at = at0 , it is easy to recover canonical averages using standard unbiasing
procedures:34 although
the adaptive biasing force dynamics has been applied so
far almost exclusively to chemical and biological systems , it could
also be very useful for sampling a multimodal measure ( namely , a
probability measure for which high - probability regions , called modes
in this context , are separated by low - probability regions ) in other
fields , e.g. , among others , free - energy computations in material sciences or markov chain monte carlo techniques for
bayesian inference . in summary ,
the
combination of simplicity , versatility , and strong mathematical underpinnings
makes the adaptive biasing force method an attractive target for a
wide variety of extensions in statistical mechanics and beyond . | in the host of numerical schemes
devised to calculate free energy
differences by way of geometric transformations , the adaptive biasing
force algorithm has emerged as a promising route to map complex free - energy
landscapes .
it relies upon the simple concept that as a simulation
progresses , a continuously updated biasing force is added to the equations
of motion , such that in the long - time limit it yields a hamiltonian
devoid of an average force acting along the transition coordinate
of interest .
this means that sampling proceeds uniformly on a flat
free - energy surface , thus providing reliable free - energy estimates .
much of the appeal of the algorithm to the practitioner is in its
physically intuitive underlying ideas and the absence of any requirements
for prior knowledge about free - energy landscapes . since its inception
in 2001
, the adaptive biasing force scheme has been the subject of
considerable attention , from in - depth mathematical analysis of convergence
properties to novel developments and extensions .
the method has also
been successfully applied to many challenging problems in chemistry
and biology . in this contribution , the method is presented in a comprehensive ,
self - contained fashion , discussing with a critical eye its properties ,
applicability , and inherent limitations , as well as introducing novel
extensions . through free - energy calculations of prototypical molecular
systems ,
many methodological aspects are examined , from stratification
strategies to overcoming the so - called hidden barriers in orthogonal
space , relevant not only to the adaptive biasing force algorithm but
also to other importance - sampling schemes . on the basis of the discussions
in this paper , a number of good practices for improving the efficiency
and reliability of the computed free - energy differences
are proposed . |
food borne disease remains a significant problem in the united states , annually affecting millions of people .
for example , the latest data reported by the centers for disease control and prevention ( cdc ) reveal that in 2007 there were 1096 outbreaks and 21,201 cases throughout the united states.1 these outbreaks reflect only those data reported to the cdc and so must underestimate the total number of food related outbreaks .
in fact a study published in 1999 , not dependent exclusively on reports to the cdc , found that approximately 76 million illnesses , 325,000 hospitalizations , and 5,000 deaths occurring annually in the u.s .
are attributable to food - related diseases costing an estimated $ 6.534.9 billion.2 unfortunately , nothing has been published more recently allowing a current evaluation of the trend of food borne outbreaks in the u.s . however , existing data are sufficient to conclude that food borne disease presents an important public health problem to the nation .
the most commonly reported contributing factors for food borne disease outbreaks are poor personal hygiene of food workers , contamination of potentially hazardous foods with pathogens , leaving food at room temperatures for an extended period of time , and insufficient time and/or temperature during initial cooking or reheating.3 the cause of the majority of these outbreaks ( 88% ) are microbial pathogens contaminating food items prepared at licensed facilities , mainly from restaurants , school cafeterias , and nursing homes.3 routine inspections of food service facilities long have been employed as regulatory tools to enforce sanitary codes and reduce the risk of food borne outbreaks . 4,5 failure to meet regulatory standards are assumed to increase the risk of food borne disease .
certainly it is not uncommon to find food borne outbreaks associated with facilities with a history of regulatory failure.6 poor food safety practice also has been identified as one of the reasons for an increase in food borne disease outbreaks found in schools , colleges , and universities in the u.s.7 in addition to enforcing regulatory inspection programs , many health departments try to ensure appropriate food safety practices through the use of mandatory or voluntary food safety and hygiene training and certification programs for food personnel .
however , the literature is inconclusive about the effectiveness of such training programs for improving food safety and protecting consumer health .
evaluated the effectiveness of a food manager training and certification program to increase compliance with sanitary codes in boston.8 their study clearly showed a significant improvement in mean inspection scores in restaurants after training managers compared to restaurants not receiving this training .
they also found a significant decrease in critical violations in restaurants with trained managers compared to restaurants without trained managers one year after training , but after two years there were deficiencies in some critical food safety elements in both types of restaurants .
reported that restaurants in toledo , ohio with trained and certified food mangers had significantly higher visual inspections scores than restaurants without trained and certified food managers .
however , there were no significant differences in the presence of fecal bacteria on food contact surfaces as a function of manager training and certification.9 hammond et al .
compared the number of food borne outbreaks and the number of cases by contributing factors before and after training was mandated in florida .
although the overall number of food borne outbreaks associated with contributing environmental factors declined significantly after the training , a significant increase in violations was found in 59% of the contributing - factor categories.10 in a literature review of the effectiveness of public health intervention in food safety in canada and elsewhere , campbell et al.11 found some studies reporting improvement in inspection scores and in food safety knowledge after food safety training and certification , and others not finding such improvements after similar training and certification .
a more current review of studies also found that the reported effectiveness of training and certification in improving inspection scores was inconsistent among studies.12 similarly , mixed results have been reported in other studies conducted outside of the united states .
one study conducted in ireland concluded that better general formal training in food safety and hygiene practices was not significantly linked to better food safety practices.13 two studies conducted in the united kingdom also concluded that food safety training and certification did not significantly improve hygiene standards of food premises.14,15 however , a study in canada and another in the united kingdom reported improvement in food safety following training and certification of food handlers.16,17 a paper published in 2009 also found restaurants with certified kitchen managers had significantly reduced critical violations compared to restaurants without certified kitchen managers.18 clearly , it is important to know if manager training and certification decrease the risk of food borne disease . if certification is not an effective tool , its use would be wasteful and may provide a false sense of security . yet , recommending against such programs might be viewed as a draconian response to an uncertain situation . before the cessation of such programs
could be recommended , there would need to be great assurance that these programs were not helpful . to address this issue we conducted a large - scale study comparing the sanitary code violations of restaurants with and without trained and certified food service facility managers .
our results should add to the literature by examining specific food code violations and selected food service facility attributes with respect to training and certification .
ultimately , we hope that our results help shed additional insight regarding the value , or the wastefulness , of food service facility manager training programs .
thus , our overall objective is to help guide the use of certification and training programs to help minimize the risk of food borne disease .
in this study , we examined food inspection reports from the toledo / lucas county health department ( ohio ) from the one - year period march 2005 through february 2006 to explore the relationship between certification training of personnel and food safety violations .
we focused on those types of facilities offering the highest level of risk , further separated into restaurant and institutional categories .
institutional facilities included hospital cafeterias , nursing home cafeterias , school cafeterias , daycare facilities , workplace cafeterias and similar establishments .
grocery stores and carry - outs were not included in this study . in ohio ,
registered sanitarians conduct walk - through inspections of licensed food service facilities for compliance with sanitary codes.19 following each inspection , a standard inspection report is provided to the facility manager documenting written violations and allowing for additional comments .
the required inspection frequency of each facility is based on a risk categorization of food service operation .
there are four licensing categories , ranging from category one ( low risk - operations ) to category four ( high - risk operations ) .
category four food service facilities prepare and serve food to high risk clienteles and/or reheat and serve bulk quantities of cooled or leftover foods .
we limited our review to category four food service operations because this group presents the highest risk for food borne outbreaks .
category four facilities are inspected a minimum of four times each year ; two - standard inspections and two critical control point inspections ( ccp ) .
food service operations may be re - inspected as often as necessary to ensure compliance with inspection orders to correct violations .
additionally , they may be inspected to investigate consumer complaints , particularly if the complaint item is a critical food - safety element with a substantial potential to cause or contribute to a food borne disease outbreak .
critical violations carry a higher risk of food borne illness than do non - critical violations . in this study
, we defined any violation in any of these four categories as critical : ( 1 ) time and temperature , ( 2 ) poor hygiene practices by food handlers , ( 3 ) cross - contamination , and ( 4 ) food from unapproved sources .
these non - critical violations do not present any immediate threat to food safety , and include such items as lack of cleanliness of non - food contact surfaces , inappropriate handling of facility wastes , and the use of non - industrial grade equipment .
we compared results , using t - tests for equality of means to establish statistical significance , from category four facilities employing personnel who had been certified through coursework in food safety and hygiene with facilities without certified employees .
we distinguished between facilities on the basis of at least one person ( usually the manager , but occasionally a chef or a food handler ) having been certified as successfully completing a food - safety training course from a provider recognized by the ohio department of health .
anova was used to determine if differences existed between the number and types of violations as a function of a food service facility being part of a larger group of facilities .
we examined 1034 inspection reports from 605 food service facilities . 26 percent of the inspections were of facilities with certified personnel , the remaining 74 percent of the facility inspections had no personnel who had received training for certification .
food service facilities with certified personnel had significantly fewer critical violations than food service facilities without certified personnel ( p 0.05 ) .
the mean number of critical violations per inspection was 1.75 for food service facilities with certified personnel and 2.08 for food service facilities without certified personnel . in comparison , food service facilities with certified personnel had higher , although not statistically significant ( p 0.05 ) non - critical violations than food service facilities without certified personnel .
the mean number of non - critical violations was 3.97 for food service facilities with certified personnel and 3.60 for food service facilities without certified personnel ( table 1 ) .
when separating the data from all facilities into two categories , restaurants and institutional , the pattern remained that those restaurants with certified personnel had fewer critical violations and more non - critical violations than restaurants without certified personnel ( table 2 ) .
the significance of this pattern changed , with certification linked to significantly more non- critical violations but not significantly fewer critical violations ( p 0.05 ) .
this change may not be very meaningful however , and simply reflect p values very close to 0.05 .
in institutional facilities , no difference was evident between facilities with and without certified personnel with regard to either critical or non - critical violations ( table 3 ) .
institutional facilities had a significantly lower number of total violations , critical violations and non - critical violations than restaurants ( p 0.001 ) ( table 4 ) .
furthermore , institutional facilities with certified personnel had significantly fewer of all types of violations than restaurants with certified personnel , a difference that was also significant for institutions without certified personnel ( table 5 ) .
the number of total violations , critical violations , and non - critical violations outlets of a food service facility varied significantly as a function of the number of outlets of that particular food service facility chain ( p 0.001 ) . in general ,
restaurants with many individual outlets had a smaller number of violations than restaurants operating individually or with only a few facilities in the chain ( table 6 ) .
this trend is particularly obvious in comparing the food service facilities with the fewest outlets ( e.g. 17 facilities ) to those with the greatest number of outlets ( e.g. 10 to 16 facilities ) . the majority of critical violations were in code category 3 , with most of these violations being in the categories of time and temperature and cross - contamination of foods during storage and preparation .
food code category 4 was next in importance , with most violations being due to lack of sanitation of equipment and utensils ( table 7 ) .
meaningful differences were not evident with regard to category of violation as a function of certification .
the largest number of non - critical violations was due to problems with operation and maintenance of equipment .
code category 6 was next in importance , with the most numerous violations being due to problems with maintenance and operations of facilities ( such as cleanliness of non - food contact surfaces , and unapproved equipment ) ( table 6 ) .
again the pattern of violation between facilities with and without certified personnel did not vary in any meaningful way .
certification in our study area did not have a major effect on the total number of violations .
the distribution of critical and non - critical violation varied as a function of the presence of certified personnel .
restaurants with certified personnel had about the same amount fewer critical violations as they had greater non - critical violations , as compared to restaurants without certified employees .
it is difficult to envision why certification would lead to an increase in non - critical violations , so suspicion falls on the inspection process .
inspections can not all be done in an identical way , and inspectors have considerable latitude in deciding upon an appropriate level of effort .
one indicator of inspection completeness ( consciously or unconsciously ) may be that a sufficiently number of violations has been found .
if certification does lead to better food handling practices with fewer critical violations , inspectors may not feel satisfied with their effort until they have identified a sufficient number of violations .
thus , one possible explanation for these data is that a larger percentage of the non - critical violations possible to identify during an inspection are identified for those restaurants for which fewer critical violations are evident . to test this hypothesis
would be difficult , as it may reflect simply a sense of accomplishing the given task on the part of individual inspectors .
another possibility that might account for these differences is that certified personnel may be focusing on critical violations to such an extent that they care less about non - critical violations .
perhaps the non - certified personnel give equal attention to all food code issues , while the certified personnel make a decision ( consciously or unconsciously ) that their time is best spent minimizing critical violations , and so do not spend as much time preventing non - critical violations .
although the number of critical violations and non - critical violations differed as a function of having certified personnel , the relative amount of specific critical violations and non - critical violations did not change .
for example , violations of food temperature , safety , contamination and on premises labeling ( category 3 ) constituted about two thirds of the critical violations in facilities with , and without , certified personnel .
similarly , violations of equipment , utensils and linen ( category 4 ) violations were the predominant non - critical violations in both types of facility .
the reason why certification did not have a major effect on the total number of violation also may be related to the type of food safety training .
the type of training that the majority of the certified personnel in this study received focused on theoretical concepts of food safety with little or no emphasis on a hands - on food safety training approach .
the importance of a hands - on approach was shown in comparing a hands - on approach in addition to lecture only approach in food safety training .
food handlers that received hands - on interactive training in addition to lecture / video presentation had statistically better test performances after the training and two weeks later than those that only received a lecture / video presentation . 20 in addition ,
training alone may not be sufficient unless it changes trainees attitudes and subsequently their behaviors in food safety practices .
the attitude of trainees and their perception of the value of compliance might influence whether or not they change their past food safety and hygiene practice behavior.21 although restaurants showed a pattern of certification affecting their number of violations , this pattern was not detectable in institutional facilities .
reasons for this difference are not apparent from analysis of the data , and can only be conjectured .
one reason may be that individual restaurants have a different objective than institutional facilities with the former being profit and the latter being to provide a service .
of course this is an over - simplification , but nevertheless may have sufficient validity to be expressed in having a different level of incentive to comply with food service regulations .
those restaurants that have invested in certified personnel may have made that investment because they care more about food safety , and thus have fewer critical violations .
institutional ( non - restaurants ) have a substantially and significantly lower number of violations ( both critical and non - critical ) than restaurants
it is reasonable to assume that institutional settings are driven more by food safety concerns than are restaurants .
interesting to note is that restaurants with certified personnel had substantially more violations than institutional facilities without certified personnel .
we do not know the reason for this , but it may be related to the customers they serve .
hospital , nursing home , and school cafeterias , daycare facilities prepare food to serve high risk clienteles for food borne diseases , although people with lower risk also consume food prepared in these facilities .
host biological factors such as age or presence of other underlying diseases and environmental factors such as being in semi - closed environments for extended periods of time increases the likelihood of secondary food borne disease transmission .
perhaps food managers and administrators of these institutions , being aware of the high risk of their clients for food borne diseases , take extra care to implement food safety guidelines .
a number of these franchised or chain restaurants have their own internal food inspectors and guidelines consistent with or more stringent than food safety regulations of local board of health .
many fast - food type restaurants have very standardized practices , often using specialized equipment that minimizes opportunity for user error .
this standardization , along with increased training and self - inspections , may account for those facilities with many outlets averaging significantly fewer violations that facilities with few outlets ( see table 5 ) .
interesting future work would be to try to decouple the importance of standardized operation from training and inspections in improving food safety .
perhaps the most important finding of this work is that training programs appear to affect most meaningfully those restaurants that are not part of chains or large franchises .
institutional food service facilities and restaurants that are part of a larger unit regularly perform better than individual restaurants , as measured by failure to comply with regulatory food codes .
this finding may prove useful to local communities efforts to improve food sanitation may be most productively directed toward individual restaurants ( or those with few companion restaurants ) .
of course , these findings do not directly predict the behavior of an individual facility , but do suggest where future efforts would be most productive .
these findings also suggest that regulatory programs requiring uniform inspections of all type of facilities may be improved through directing efforts more toward non - institutional or chain restaurants .
finally , those characteristics of institutional and chain restaurants that result in fewer violations should be identified in future research , and efforts made to apply this knowledge at the level of individual restaurants . | food safety is an important public health issue in the u.s . eating at restaurants and other food service facilities
increasingly has been associated with food borne disease outbreaks .
food safety training and certification of food mangers has been used as a method for reducing food safety violations at food service facilities . however , the literature is inconclusive about the effectiveness of such training programs for improving food safety and protecting consumer health .
the purpose of this study was to examine the effect of food manger training on reducing food safety violations .
we examined food inspection reports from the toledo / lucas county health department ( ohio ) from march 2005 through february 2006 and compared food hygiene violations between food service facilities with certified and without certified food managers .
we also examined the impact on food safety of a food service facility being part of a larger group of facilities.restaurants with trained and certified food managers had significantly fewer critical food safety violations but more non - critical violations than restaurants without certified personnel .
institutional food service facilities had significantly fewer violations than restaurants , and the number of violations did not differ as a function of certification .
similarly , restaurants with many outlets had significantly fewer violations than restaurants with fewer outlets , and training was not associated with lower numbers of violations from restaurants with many outlets . the value of having certified personnel was only observed in independent restaurants and those with few branches .
this information may be useful in indicating where food safety problems are most likely to occur .
furthermore , we recommend that those characteristics of institutional and chain restaurants that result in fewer violations should be identified in future research , and efforts made to apply this knowledge at the level of individual restaurants . |
arthropods ( including insects and arachnids ) are often defined as pests , because they either compete with human consumption in agricultural or urban settings or transmit causal pathogens of disease .
a large amount of investment is made into development of control tools for such pests .
these tools can vary widely and significant progress has been made in developing biorational tools that either modify or exploit the natural behavior of pests with the use of behavioral attractants or repellents .
. therefore discovery of effective attractants or repellents for pests can often lead to development of control tools .
for example , one must ascertain whether one sex of a given species attracts the opposite sex for mate location .
if so , one must determine whether this attraction is mediated by a chemical and where that chemical is produced .
further steps include determining when the chemical is produced and its eventual identification . throughout this procedure ,
the test subject 's ( insect or mite ) behavioral response to odorants must be tested .
initially , the behavior of the test subject will be tested to the natural material .
for example , a male moth 's response may be tested to that of the extract of its female conspecific 's pheromone gland .
alternatively , an herbivore 's response to the natural odors of its host plant may be tested .
while these investigations often have a practical focus on development of management tools , they can also be applied to research that addresses more fundamental questions , such as speciation .
subsequent steps involve testing the arthropod 's response to synthetic versions of identified natural products .
understanding how arthropods respond to chemicals requires the development of appropriate tools to measure their behavior .
the data generated by such tools will depend on how well the investigator is able to measure natural behaviors in a controlled setting . over six decades ago , sustained flight olfactometers and tunnels were developed to measure insect response to odorants .
olfactometers vary significantly in design and are often tailored to the specific insect or problem being investigated .
the goal of this article is to describe the design and use of a vertically oriented , t - maze olfactometer for measuring insect response to odorants ( figure 1 ) .
the olfactometer 's vertical orientation takes advantage of the negative geotaxis ( tendency to move up ) exhibited by many insects . a light source positioned directly above
the olfactometer takes advantage of the positive phototaxis ( tendency to move toward source of light ) exhibited by many insects .
the olfactometer is particularly useful for hemipteran insects that tend not to exhibit movement readily in horizontally - oriented olfactometers and that are heavily influenced by light intensity with respect to orientation .
however , this device can be used for a wide range of flying and crawling insects , as well as arachnids .
the olfactometer can be employed to evaluate both attractants and repellents . in the case of attractants , the bifurcated odor field ( figure 1 )
is established with the putative attractant released on one side versus clean air released on the opposite side .
a choice is recorded when the insect released at the base of the olfactometer chooses one of the two odor fields . in the case of repellents ,
in addition to upward movement into either of the two odor fields , lack of upward movement may be recorded indicating a non - responsive stimulus or repelling stimulus from either odor fields .
assembly of a custom - designed , two - port divided t - olfactometer previously described in mann et al . is shown in figure 1 .
the olfactometer consists of a 30 cm glass tube that is bifurcated into two separate ports with a teflon strip forming a t - maze .
each half is analogous to an arm of a 2-choice style olfactometer enabling the test subject to choose between two potential odor fields .
assembly and subsequent assays should take place within a temperature - controlled room or environmental chamber.mount glass t - maze olfactometer vertically as shown in figure 1 and position it under a fluorescent 900 lux light bulb.for optimal light diffusion , the assembly should be mounted within a 1.0 x 0.6 x 0.6 m opaque container to maximize uniform light diffusion.connect the olfactometer arms to odor sources that will be housed in solid - phase micro - extraction holding / odor source chamber ( spmec ) ( ars , inc . ,
each spmec is comprised of a straight glass tube ( 17.5 cm long x 3.5 cm wide ) supported with an inlet and outlet valve for incoming and outgoing air streams ( figure 1).connect the air delivery system to an external source of humidified and carbon - purified air that is under constant pressure ( the air delivery system has both an internal carbon filter for air purification and an air bubbler for humidification if external sources are unavailable).dissolve desired chemical samples within appropriate diluent ( solvent ) and pipette onto a slow release substrate such as a cotton wick ( petty john packaging , inc . , concord , nc ) .
the control treatment should consist of a cotton wick impregnated with solvent only.to prevent significant contamination of the glassware , envelop the treated and control cotton wicks in laboratory tissue ( kimwipes , kimberly - clark , roswell , ga ) and place into the two spmec arms ( ars , inc . , gainesville , fl ) . in the case of live plant samples
, a guillotine volatile collection chamber can be attached to the olfactometer containing treatments in place of the spmec ( figure 1).deliver purified and humidified air through the spmec ( or guillotine volatile chamber ) via two pumps connected to an air delivery system ( figure 1 ) .
maintain airflow rate at 0.1 l / min through both arms of the olfactometer .
assembly and subsequent assays should take place within a temperature - controlled room or environmental chamber .
mount glass t - maze olfactometer vertically as shown in figure 1 and position it under a fluorescent 900 lux light bulb . for optimal light diffusion
, the assembly should be mounted within a 1.0 x 0.6 x 0.6 m opaque container to maximize uniform light diffusion .
connect the olfactometer arms to odor sources that will be housed in solid - phase micro - extraction holding / odor source chamber ( spmec ) ( ars , inc . ,
each spmec is comprised of a straight glass tube ( 17.5 cm long x 3.5 cm wide ) supported with an inlet and outlet valve for incoming and outgoing air streams ( figure 1 ) . connect the air delivery system to an external source of humidified and carbon - purified air that is under constant pressure ( the air delivery system has both an internal carbon filter for air purification and an air bubbler for humidification if external sources are unavailable ) .
dissolve desired chemical samples within appropriate diluent ( solvent ) and pipette onto a slow release substrate such as a cotton wick ( petty john packaging , inc .
the control treatment should consist of a cotton wick impregnated with solvent only . to prevent significant contamination of the glassware , envelop the treated and control cotton wicks in laboratory tissue ( kimwipes , kimberly - clark , roswell , ga ) and place into the two spmec arms ( ars , inc . ,
, a guillotine volatile collection chamber can be attached to the olfactometer containing treatments in place of the spmec ( figure 1 ) .
deliver purified and humidified air through the spmec ( or guillotine volatile chamber ) via two pumps connected to an air delivery system ( figure 1 ) .
employing olfactometer to test arthropod response to putative repellents . in the present description , a hemipteran insect , asian citrus psyllid , diaphorina citri ,
response was tested to citrus volatiles alone or in combination with a repellent ( dimethyl disulfide ) and clean air as odor sources .
conduct all experiments at a standardized temperature and relative humidity.prior to all tests , expose test subjects to clean air versus clean air or solvent versus clean air in the t - maze olfactometer to verify the absence of positional bias or an effect of solvent on behavior of the test subject.assign an odor source randomly to one of the arms of the olfactometer at the beginning of each bioassay .
reverse this position after every 10 test subjects assayed , at minimum , to eliminate potential of positional bias.test the response of a minimum of 30 ( and up to 120 ) test subjects per treatment combination.allow test subjects 100 - 300 sec to exhibit a behavioral response based on previously established response ( latency ) interval.record if subject enters the treatment arm , control arm or remains in the release port or below the t - maze division.score a treatment or control arm choice when a test subject crosses the division in the t - maze and moves into either olfactometer arm.score a release arm choice when a test subject remains in the release port or below the t - maze division.clean the olfactometer and connecting tubes thoroughly with 2% soap solution and bake the glass components at 200 f between the use of different odor treatments.for assays in which putative repellent treatments are presented in the t - maze olfactometer with or without a chemical treatment versus clean air , compare the number of test subjects remaining at the release point and not entering the olfactometer between treatments by one way analysis of variance ( anova ) followed by tukey 's hsd test ( < 0.05 ) . in cases
in which the test subjects leave the release arm , compare the number choosing the control arm versus the treatment arm with chi square ( ) analysis at < 0.05 .
prior to all tests , expose test subjects to clean air versus clean air or solvent versus clean air in the t - maze olfactometer to verify the absence of positional bias or an effect of solvent on behavior of the test subject .
assign an odor source randomly to one of the arms of the olfactometer at the beginning of each bioassay .
reverse this position after every 10 test subjects assayed , at minimum , to eliminate potential of positional bias .
test the response of a minimum of 30 ( and up to 120 ) test subjects per treatment combination .
allow test subjects 100 - 300 sec to exhibit a behavioral response based on previously established response ( latency ) interval .
record if subject enters the treatment arm , control arm or remains in the release port or below the t - maze division .
score a treatment or control arm choice when a test subject crosses the division in the t - maze and moves into either olfactometer arm .
score a release arm choice when a test subject remains in the release port or below the t - maze division .
clean the olfactometer and connecting tubes thoroughly with 2% soap solution and bake the glass components at 200 f between the use of different odor treatments . for assays in which putative repellent treatments are presented in the t - maze olfactometer with or without a chemical treatment versus clean air , compare the number of test subjects remaining at the release point and not entering the olfactometer between treatments by one way analysis of variance ( anova ) followed by tukey 's hsd test ( < 0.05 ) . in cases
in which the test subjects leave the release arm , compare the number choosing the control arm versus the treatment arm with chi square ( ) analysis at < 0.05 .
attraction of asian citrus psyllid ( diaphorina citri ) adults to the odors of their natal host plant volatiles ( citrus ) is depicted in figure 2a .
significantly ( < 0.05 ) more adults chose the arm of the t - maze olfactometer receiving odors from living citrus plants as compared with a blank ( clean air ) control .
an example of repulsion is shown in figure 2b . in this case , the insects were exposed to one arm of the t - maze receiving citrus volatiles , while the second arm received volatiles from a citrus plant that was treated with the known repellent , dimethyl disulfide . in this case , three types of behaviors were observed .
the number of psyllids arrested at the release arm and not responding did not differ statistically from the number entering the arm receiving the odors from citrus plants alone ( figure 2b ) .
however , no psyllids entered the arm containing the host plant volatile when co - presented with the repellent ( figure 2b ) .
schematic diagram of vertically oriented t - maze olfactometer joined to air delivery system and odor treatment release devices .
the diagram is an adaptation of figure 1 in mann et al .. a test subject is placed individually into the release chamber of the 2-port divided t - olfactometer .
it moves towards the odors through a gate valve which allows controlled upward movement into the olfactometer and prevents the insect from crawling back into the release point area .
the tube bifurcates 10 cm after the gate value . a positive response to an odor
is recorded once the insect moves 0.5 cm into a specific side of the division .
the guillotine volatile collection chamber may be used in place of a solid - phase micro - extraction holding / odor source chamber ( spmec ) as an odor source .
response of asian citrus psyllid ( diaphorina citri ) adults to volatiles from citrus host plants versus clean air control ( a ) or citrus volatiles versus citrus volatiles co - released with a repellent ( dimethyl disulfide ) ( b ) in a vertically oriented t - maze olfactometer .
figure 2a : one - way analysis of variance ( anova ) followed by tukey 's hsd test ( < 0.05 ) was performed to compare the number of psyllid making a choice between either arm ( 2 treatments , n = 120 ) .
columns indicated by different letters are significantly different from one another ( < 0.05 ) .
figure 2b : one - way analysis of variance ( anova ) followed by tukey 's hsd test ( < 0.05 ) was performed to compare the number of psyllid making a choice between either arm or remaining in the release point ( 3 treatments , n = 120 ) .
columns indicated by different letters are significantly different from one another ( < 0.05 ) .
herein , an assay device and protocol are described for measuring the response of small arthropods ( insecta : hemiptera : psyllidae ) to odorants .
the method involves a choice test , allowing the insect to make a choice between two odorant fields in the case of assays evaluating a putative attractant .
furthermore , the test subject may display three types of behaviors by leaving the release arm and entering either one of two potential odor fields , or remaining in the release arm , in the case of repellency assays .
the olfactometer allows for high throughput data collection because it takes advantage of the negative geotactic ( tendency to move upward ) and positive phototactic ( tendency to move toward light ) natural behavioral response of many arthropods .
although the present demonstration uses a psyllid insect test example , the assay can be readily adapted to arthropods broadly , both those that use flight or walking as predominant modes of transportation .
olfactometers designed for measuring the response of insects were initially developed decades ago and have played a major role in elucidating insect attractants and repellents .
the specific designs of such olfactometers vary widely ; however , variations on the general theme of the two - choice assay described here , as well as , similar y - tube assays have been often used to measure arthropod response to chemicals .
larger flight tunnels that may cause sustained flight of insects have also resulted in collection of groundbreaking data elucidating the fundamental mechanisms of insect flight and orientation to semiochemicals , as well as , data informing practical use of pest control tools .
it is often necessary to tailor design an olfactometer and associated instrumentation for the biology of the specific arthropod test subject .
those olfactometers that can be used among a general group of insects are more useful than ones specific to a small group ; however , sometimes the economic significance of a small group of insects dictates the need for development of a very specific olfactometer and assay technique .
it allows for a more standard two - choice assay than y - tube bioassays in which the experimental arena or choice test takes the form of a y - shaped glass device .
typically , one arm of such a y - tube will receive a treatment odorant , while the other will be left blank .
variations on such olfactometers may include addition of multiple radiating arms and even addition of a soil medium for assaying the behavior of organisms moving through soil . in developing such olfactometers and associated assays ,
it is important to consider how closely natural conditions are replicated and thus the true relevance of the assessed behavioral responses in terms of the test subjects ' biology . to a large degree
, the data collected will only be as useful as the relevance of the behavioral bioassay with respect to the organism 's behavioral ecology .
the currently described olfactometer and behavioral bioassay is specifically designed for a hemipteran insect that tends to initiate flight as short " jumps " .
the vertical orientation of the olfactometer and the light source arrangement facilitate initiation of insect movement and thus subsequent chemically - mediated orientation behavior can be assayed efficiently and with high throughput .
this behavioral assay arrangement can likely be used for a broad array of flying or walking arthropod taxa or could be also easily modified to fit the need of non - arthropod organisms .
| given the economic importance of insects and arachnids as pests of agricultural crops , urban environments or as vectors of plant and human diseases , various technologies are being developed as control tools .
a subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion .
therefore , arthropods are often the focus of behavioral investigations .
various tools have been developed to measure arthropod behavior , including wind tunnels , flight mills , servospheres , and various types of olfactometers .
the purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues .
the vertical t - maze oflactometer described here measures choices performed by insects in response to attractants or repellents .
it is a high throughput assay device that takes advantage of the positive phototaxis ( attraction to light ) and negative geotaxis ( tendency to walk or fly upward ) exhibited by many arthropods .
the olfactometer consists of a 30 cm glass tube that is divided in half with a teflon strip forming a t - maze .
each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants . in assays involving repellents ,
lack of normal response to known attractants can also be measured as a third variable . |
various modalities , such as percutaneous nephrolithotomy ( pcnl ) , extracorporeal shock wave lithotripsy , and retrograde intrarenal surgery , are used to manage renal stone disease [ 1 - 3 ] . however , pcnl is regarded as the treatment of choice for most renal stones , especially for patients with a large renal stone ( > 2 cm ) , an infected stone , or a cystine stone and for those with a history of failed shock wave lithotripsy .
general anesthesia during pcnl has many advantages , for example , it enables breathing control and improves patient comfort .
however , associated complications and cost are higher for general anesthesia than for regional anesthesia . for example
, endotracheal tube migration and neurologic problems , particularly at the time of position transition , may arise during pcnl under general anesthesia .
unfortunately , few research studies have been conducted to compare regional and general anesthesia with respect to operative parameters . in the present study , therefore , to determine whether pcnl under regional anesthesia is a better alternative than pcnl under general anesthesia , we examined surgical outcomes after these two procedures .
one hundred and one consecutive patients who underwent pcnl as a first - line management for renal stones , including staghorn , pelvic , and calyceal stones , from june 2004 to june 2013 at inje university sanggye paik hospital were included in this retrospective study .
the patients were classified into two groups by anesthesia method : a regional anesthesia group ( n=77 ) and a general anesthesia group ( n=24 ) .
decisions regarding anesthesia method were mainly based on considerations of medical status , such as cardiac problems and the presence of respiratory illness or coagulopathy . in the absence of such factors ,
the contraindications for pcnl were uncorrected coagulopathy , congenital renal anomaly , and metastatic malignancy .
in addition to history taking , we performed a physical examination , preoperative laboratory tests , and a radiological evaluation , which included plain radiography of the kidney - ureter - bladder ( kub ) region and abdominopelvic computed tomography ( ct ) .
stone size was defined as the sum of the maximal lengths of renal stones on ct images .
patients with a urinary tract infection were treated preoperatively in accordance with urine culture sensitivity .
the clinical and stone parameters compared were age , gender , body mass index , stone laterality , stone size , and stone number , respectively .
regional anesthesia was induced with 0.5% heavy bupivacaine ( marcaine , astrazeneca korea , seoul , korea ) 12 ml , and a spinal epidural set ( portex , smiths medical , kent , uk ) was injected intrathecally into the l3 - 4 or l4 - 5 interspaces .
the level of anesthesia was checked for 5 minutes after injection with the patient in the trendelenburg position .
if surgery was prolonged , levobupivacaine ( chirocaine , abbott korea , seoul , korea ) was administered by an anesthesiologist to maintain adequate anesthesia .
general anesthesia was carried out by using intravenous propofol ( pofol , dongkook pharmaceutical co. , seoul , korea ) and rocuronium ( esmeron , msd korea , seoul , korea ) . following the induction of anesthesia
an occlusion ureteral catheter was then inserted into the renal pelvis and ballooning ( 1 ml ) was performed to prevent stone fragments from descending into the proximal ureter .
patients were then rotated to the prone position and percutaneous nephrostomy was performed under ultrasonographic guidance .
the access tract was dilated with an amplatz and a balloon dilator , and stone fragmentation was performed by using a combination of ultrasonic and pneumatic lithotripsy .
stone fragments were removed by using grasping forceps under a 24-fr nephroscope . a 20-fr nephrostomy tube was left and a fluoroscopic nephrostogram was routinely obtained to assess status at the end of the procedure .
patients in both groups were radiologically followed up at 1 day ( initial ) and 1 month ( overall ) after surgery to assess status .
stone - free status was defined as the absence of any fragment greater than 4 mm by kub .
operative outcomes , surgical times , hospital stays , stone - free rates , perioperative complications , and changes in hemoglobin and white blood cell counts were compared by using the modified clavien grading system , which was proposed to grade perioperative complications of general surgery and has been validated in a cohort of 6,336 patients .
fever was defined as a temperature exceeding 37.7 at 1,600 on the first postoperative day .
postoperative antipyretics ( arthagyl , ilyang pharmaceutical co. , seoul , korea ) , analgesics ( tridol , yuhan co. , seoul , korea ) , antiemetics ( mexolon , donga pharm , seoul , korea ) , and diuretics ( lasix , handok inc . ,
seoul , korea ) were injected intravenously when symptoms appeared during the operative or first postoperative day .
blood transfusions were done in the operating room on the basis of decisions made by anesthesiologists .
chicago , il , usa ) . because the number of patients in the general anesthesia group was small ( n=24 ) , continuous variables were assessed by using the mann - whitney test , and categorical variables were analyzed by using the chi - square test .
clinical parameters are expressed as meanstandard deviations , and statistical significance was accepted for p - values < 0.05 .
patient and stone characteristics in the regional and general anesthesia groups are compared in table 1 .
mean ages in these groups were 54.812.2 years and 50.817.8 years , respectively ( p=0.338 ) .
gender and body mass index were nonsignificantly different , and mean stone sizes ( 34.524.0 mm and 42.336.1 mm , respectively ; p=0.309 ) , mean stone numbers ( 2.02.4 and 2.95.1 , respectively ; p=0.648 ) , and the distributions of renal stones were similar ( p=0.254 ) .
intergroup differences between initial stone - free rates ( 68.8% and 41.7% , respectively ; p=0.246 ) and overall stone - free rates ( 80.5% and 66.7% , respectively ; p=0.371 ) were nonsignificantly different .
no significant intergroup differences were found between mean surgical times ( 14372 minutes and 151112 minutes , respectively ; p=0.185 ) or changes in hemoglobin levels ( -1.61.2 g / dl and -1.71.3 g / dl , respectively ; p=0.734 ) in the regional and general anesthesia groups .
furthermore , white blood cell levels were not significantly changed in the two groups ( 3,8833,110/l and 3,9103,509/l , respectively ; p=0.480 ) .
however , mean hospital stay was significantly shorter in the regional anesthesia group ( 8.93.2 days vs. 11.56.9 days , respectively ; p=0.025 ) .
perioperative complications were investigated by using the modified clavien grading system , and the results are summarized in table 2 . a smaller proportion of patients in the regional anesthesia group experienced postoperative fever ( p=0.007 ) .
however , mean dosages of antipyretics ( p=0.397 ) , analgesics ( p=0.800 ) , antiemetics ( p=0.179 ) , and diuretics ( p=0.737 ) administered were not significantly different .
furthermore , the mean number of pints of blood transfused per patient was similar in the two groups ( 0.30.9 vs. 0.30.7 , respectively ; p=0.811 ) .
no patient experienced a grade iii or iv complication according to the modified clavien grading system .
pcnl remains the first - line treatment of choice for managing renal stone disease , although minimally invasive modalities , such as retrograde intrarenal surgery , have been introduced .
furthermore , most urologists prefer general anesthesia for pcnl owing to the high level of anesthesia achieved , the ability to control the patient 's breathing , and because it is more comfortable for patients . however , general anesthesia is more likely to cause severe morbidities , such as drug - induced anaphylaxis , complications associated with endotracheal tube insertion , and cardiovascular , pulmonary , and neurologic complications , than is regional anesthesia .
studies have been conducted to demonstrate the benefits of regional anesthesia in other types of surgery , such as radical retropubic prostatectomy and total hip arthroplasty .
salonia et al . asserted that regional anesthesia permits fine muscle relaxation and achieves excellent surgical outcomes after radical retropubic prostatectomy .
furthermore , regional anesthesia has been reported to reduce the risk of intraoperative hemorrhage , to be associated with less postoperative pain , and to allow earlier restoration than general anesthesia [ 18 - 20 ] .
in addition , gonano et al . reported that the cost of regional anesthesia is 40% less than that of general anesthesia during orthopedic surgeries .
several comparative studies on anesthesia methods in pcnl have demonstrated various benefits for pcnl under regional anesthesia compared with general anesthesia . in a retrospective comparative study of 37 patients who underwent regional anesthesia and 45 who underwent general anesthesia ,
it was concluded that the results were comparable in terms of general profiles , operative times , and stone - free rates . in another study of 50 patients who underwent pcnl ,
regional anesthesia was found to be associated with greater patient satisfaction , less early postoperative pain , and fewer adverse events than general anesthesia .
furthermore , in a prospective randomized study on pcnl in 64 patients ( 32 general and 32 regional anesthesia ) , patients in the regional anesthesia group were found to have significantly lower postoperative analgesic demands and shorter hospitalization periods .
reported that general anesthesia increases the probabilities of fluid absorption and electrolyte imbalance , and other authors have recommended regional anesthesia for patients with an electrolyte imbalance , especially for morbidly obese patients . despite the opinion held by some that regional anesthesia is unsuitable for pcnl of calculi in the upper pole of the kidney , regional anesthesia ( bupivacaine [ marcaine ] , 12 - 15 mg ) can be used to anesthetize up to the t4 level ( level of axilla ) according to an anesthesiology textbook .
general anesthesia is associated with significant changes in the shape of the chest and alveolar gas contents .
outside of that , these changes result in continued elevation of the risk for reintubation , mechanical ventilation , and nosocomial pneumonia , all of which prolong the hospital stay .
regarding hospital stay , it was previously reported that average hospital stay is approximately 5 to 7 days regardless of anesthesia type .
however , in the present study , hospital stays were 8.93.2 days and 11.56.9 days , respectively , which could have been due to the exclusion of time spent at the hospital before surgery and the procedure used to determine the discharge date . in addition , according to a large - scale study , initial and overall stone - free rates were 69.9% and 88.8% , respectively , and another study conducted on 610 patients found corresponding rates of 57.6% and 84.9% , respectively . in the present study , values were 68.8% and 80.5% in the regional anesthesia group and 42.9% and 71.4% in the general anesthesia group , which were relatively low and did not represent a significant intergroup difference .
we attribute these differences to the different definitions used for the stone - free rate and patient descriptive features . in brief , this retrospective study showed that despite similar patient characteristics in the two groups , postoperative fever rates and hospital stays were significantly greater in the general anesthesia group .
thus , it may be more helpful for patients to implement pcnl under regional anesthesia than general anesthesia in matters of fever control and cost of hospitalization . the present study had some limitations that deserve mention .
first , it was inherently limited by its retrospective design ; as such , lack of control of basic patient factors may have introduced bias .
second , the study was performed at a single center , and the general anesthesia group was appreciably smaller than the regional anesthesia group .
the present study showed that in terms of general characteristics and procedural success , the outcomes of pcnl conducted by use of regional or general anesthesia are similar .
nevertheless , pcnl with regional anesthesia required fewer hospital days and was associated with a lower postoperative fever rate .
accordingly , we conclude that pcnl under regional anesthesia is at least as advantageous as pcnl under general anesthesia in patients with kidney calculi . | purposeto compare surgical outcomes and complications after percutaneous nephrolithotomy ( pcnl ) under regional or general anesthesia.materials and methodsone hundred and one patients who underwent pcnl as a first - line treatment for kidney calculi between june 2004 and june 2013 were enrolled in this retrospective study .
patients were classified into two groups by anesthetic method : 77 were allocated to the regional anesthesia group and 24 to the general anesthesia group .
patient general characteristics , stone features , surgical outcomes , and complications were compared between the two groups.resultsthe two groups were similar in terms of mean age and stone size , number , and type .
furthermore , they did not differ significantly in terms of general characteristics , treatment outcomes , or complications excluding postoperative fever .
however , mean hospital stay was significantly shorter in the regional anesthesia group than in the general anesthesia group ( 8.93.2 days vs. 11.56.9 days , respectively , p=0.025 ) .
also , the postoperative fever rate was significantly higher in the general anesthesia group ( 53.2% vs. 83.3% , respectively , p=0.007).conclusionsregional anesthesia is as effective as general anesthesia during percutaneous nephrolithotomy and is associated with shorter hospital stays and lower rates of postoperative fever . |
the evaluation of motor function for duchenne muscular dystrophy ( dmd ) includes the
10-meter run , 6-minute walk test ( 6mwt ) , and the north star ambulatory assessment ( nsaa ) .
these evaluation methods have also been used to determine the appropriate time to commence
corticosteroid therapy for patients with dmd .
the reliability and validity of the 6mwt and
nsaa have been confirmed in a multi - center longitudinal study1 , and these assessment methods have been employed in clinical
studies2 .
the outcomes of these tests
can help assess an individual s motor ability in detail ; however , these tests have certain
limitations when they are performed at a hospital .
they are usually performed at the time of
the patient s visit to the hospital , which makes them less frequent and discontinuous .
moreover , the results are easily influenced by the physical or emotional condition of the
patient .
it has been reported that the amount of daily life activity for the motor - functional
evaluation of healthy children and pediatric diseases3,4,5,6 can be monitored using an
actigraph .
furthermore , the amount of activity recorded by an actigraph has been used as the
main outcome index to reflect the amount of physical activity in the home environment of
patients with dmd6 .
although , these
reports did not compare the results of actigraph monitoring with conventional motor - function
evaluation methods , their results suggest actigraph assessment of daily life activity can be
used to evaluate motor function as effectively as the conventional motor - function tests .
moreover , an actigraph can easily and non - invasively record consecutive data . in this study , whether an actigraph can be used to effectively monitor the changes in the
motor function of a patient with dmd before and after corticosteroid administration was
examined by comparing the results with conventional motor - function evaluation methods .
the subject s
body weight was 17 kg and his body mass index and serum creatine kinase were 15.1
kg / m and 24,030 u / l , respectively .
a nonsense mutation was identified in exon
21 of the patient s dystrophin gene .
the actigraph ( mtn-210 , estera , saitama , japan ) was constantly attached to the patient s
lower waist , except during bathing , during the evaluation period .
data were collected for 14
days before and 14 days after the administration of corticosteroids , until his next visit to
the hospital .
the collected data were analyzed using dedicated software ( sleepsign act ,
kissei comtec , matsumoto , japan ) .
the evaluation index was calculated by taking the mean
energy expenditure and number of steps per day .
statistical analysis was performed using the
unpaired student s t - test and pasw statistics software ( version 22.0 , spss , inc . ,
chicago ,
il , usa ) . during the 14 days before and after the administration of corticosteroids , the
patient did not participate in any high - momentum activities such as athletic events .
conventional motor - function tests , the 10-meter run , 6mwt , and nsaa , were performed by the
patient at the time of hospital visits ( once a month ) .
the difference in the results of each
test was examined before and after corticosteroid administration .
the 6mwt was performed in
two ways the way it is conventionally defined by the american thoracic society7 ( conventional 6-minute walk test ; c6mwt ) ,
and in the way described in our recent report , paced by the sound of a metronome ( periodic
sound - based 6-minute walk test ; ps6mwt)8 .
in this
test , the subject initially walked for one minute to a long - interval metronome sound , and
the distance walked was measured .
the sound interval was then gradually shortened , and the
subject walked for one minute at each of the metronome beat intervals .
the best periodic
sound was determined as that when the subject walked the longest distance in one minute .
the
subject then walked , paced by the best periodic sound , for six minutes .
since the distance
covered in the ps6mwt by both healthy subjects and those with dmd is significantly longer
than the distance in the c6mwt , the ps6mwt provides a better indication of ambulatory
potential in the evaluation of physical endurance than the c6mwt8 .
this study was approved by the institutional ethics committee of the shinshu university
school of medicine , japan ( approval number : 2,340 ) .
the study was orally explained to the
subject and his parents , and they consented to participation in the experiment .
in the assessment of the daily life activity of the dmd patient , the mean energy
expenditure before and after corticosteroid administration was 1,108.7 137.2 kcal / day and
1,246.0 127.0 kcal / day , respectively , and the difference between the two values of mean
energy expenditure was statistically significant ( p=0.024 ) . furthermore , the mean number of
steps before and after the administration were 10,081.8 2,638.7 steps / day and 12,205.0
1,695.4 steps / day , respectively , and the difference between these values was also
significant ( p=0.033 ) .
these significant increases were observed at just 14 days after the
start of corticosteroid administration ( table
1table 1.actigraph results before and after the corticosteroid administrationbefore administrationafter administrationnumber of steps ( steps / day)10,081.8 2,638.712,205.0 1,695.4energy expenditure ( kcal / day)1,108.7 137.21,246.0 127.0 * p<0.05 , unpaired student s t - test ) .
* p<0.05 , unpaired student s t - test the results of the conventional motor - function tests at the time of the patient s visit
before and after corticosteroid administration were as follows .
the time of the 10-meter run
after administration was 0.43 seconds less than that before administration ( table 2table 2.conventional motor function test results before and after the corticosteroid
administrationbefore administrationafter administrationdifference10-m run ( s)4.43.70.43c6mwt ( m)3663726ps6mwt ( m)37539318nsaa ( score)22/3429/347c6mwt : conventional 6-minute walk test ; ps6mwt : periodic sound based 6-minute walk
test ; nsaa : north star ambulatory assessment ) .
the distance of the 6mwt after administration was longer than that before
administration ( 6 m longer in the c6mwt and 18 m longer in the ps6mwt ) ( table 2 ) .
the nsaa score increased to 29 points from 22 points
( table 2 ) .
the maximum increase was seen in
the one - leg jump item of the nsaa .
the increase from a score of zero ( indicating
impossibility ) to two ( indicating the highest score ) was confirmed for both legs of the
patient .
c6mwt : conventional 6-minute walk test ; ps6mwt : periodic sound based 6-minute walk
test ; nsaa : north star ambulatory assessment
in this study , the evaluation of the amount of daily activity and conventional
motor - function tests were used to assess a patient with dmd before and after corticosteroid
administration .
the energy expenditure and number of steps taken by the patient increased
after corticosteroid administration .
daily activity measured using an actigraph and using
conventional motor - function evaluation tests indicated the same tendency . in this study
, the conventional motor - function tests were scheduled once a month , but on
one occasion , there was a gap of more than two months between the tests .
the main reasons for the low and
discontinuous frequency of evaluations were the long distance between patient s residence
and the hospital , and the unstable physical and emotional condition of the patient .
the actigraph measured data at any
place and time , which made statistical analysis possible using the data collected during a
period of just 14 days .
our results show that an actigraph can be easily and noninvasively used to monitor daily
life activity on consecutive days for the evaluation of motor activity before and after
corticosteroid administration .
furthermore , the evaluation of motor activity using an
actigraph is consistent with the results of conventional motor - function tests .
future
studies addressing this topic should include a large number of subjects and evaluate motor
functions over a longer period to determine the utility of the actigraph as a primary tool
for measuring the outcome of clinical trials and to evaluate the efficacy of therapeutic
agencies . | [ purpose ] this study evaluated the effect of corticosteroid treatment on the daily
activity of a patient with duchenne muscular dystrophy using an actigraph and examined
whether this method produces the same results as the conventional motor - function
evaluation methods .
[ subject and methods ] a patient with 5 year - old duchenne muscular
dystrophy was recruited .
an actigraph was attached to his waist to measure the energy
expenditure and the number of steps taken by him during a period of two weeks , 14 days
before and 14 days after corticosteroid administration .
the outcomes of these measurements
were compared with the results of conventional motor - function evaluation methods the 10-m
run test , 6-minute walk test , and north star ambulatory assessment on his next visit .
[ results ] the actigraph data for energy expenditure and the number of steps taken
correlated well with the results of the above - mentioned conventional motor - function tests ,
and the value of each data point improved after corticosteroid administration .
[ conclusion ] an actigraph was effectively used to non - invasively measure consecutive daily
activity for four weeks .
it was easily done and the results were consistent with
conventional motor - function evaluation methods . |
tourette syndrome ( ts ) is a childhood onset neuropsychiatric movement disorder characterized by multiple motor tics and one or more vocal / phonic tics.1,2 a duration of more than 1 year since the first tic and an onset before 18 years is also required for diagnosis.1,2 ts is associated with both simple and complex tics .
simple motor tics are restricted to a small group of muscles in the body and simple vocal tics to small sounds including throat clearing or sniffing.3 complex tics involve several muscle groups or purposeful movements including touching or sniffing objects as well as the use of words or phrases .
complex tics can include echolalia ( repeated vocalizations ) , palilalia ( repetition of words or phrases ) , echopraxia ( repeated actions ) , palipraxia ( repeating the last act ) , self - injurious behaviors , complex vocalizations ( eg , animal sounds ) , coprolalia ( swearing ) , copropraxia ( inappropriate touching ) etc.4 other associated features include non - obscene socially inappropriate behaviors and remarks , concern for symmetry and evening up , having to do things just right and forced touching etc which can be regarded as a complex tic ( mostly involuntary with little or no warning ) or an impulsion ( resulting from poor impulse control and the person may experience pleasure or gratification in some instances ) or a compulsive behavior ( preceded by an obsessional thought or a compulsive urge ) .
although only motor and vocal tics are necessary for a diagnosis of ts , the incidence of tics in the absence of other associated features and comorbidities occurs in only around 10% of cases ( pure - ts ) while the remainder have a number of associated comorbidities ( ts - plus).5,6 in this regard , ts has been commonly associated with comorbid conditions including attention deficit hyperactivity disorder ( adhd ) , obsessive compulsive disorder ( ocd ) and to a lesser extent autism spectrum disorder.3 in addition to the core clinical features that are integral to ts , a wide range of other behavioral , emotional , and psychiatric conditions have been reported to occur more commonly in ts as compared to the general population ( eg , adhd in 60% of the ts population as compared to 5%10% in the general population and ocd in 30%50% of the ts population as compared to 1%3% in the general population ) and previous studies have also suggested that increased tic severity is associated with increased psychopathology.4,711 further , ts has been to a lesser extent associated with autism spectrum disorder.3,9,11 a number of co - existent psychopathologies have also been described in ts including anxiety , depression , learning difficulties , personality disorder , impulse control , aggression , oppositional defiant disorder , and conduct disorder.6 while the occurrence of the core clinical features and associated comorbid conditions have been found to be similar irrespective of country of origin,12 highlighting the biological nature of ts , there have been some differences noted between cultures with regard to the occurrence of associated psychopathologies , suggesting complex gene
environment interactions.13,14 although ts is known in the lay literature for coprophenomena ( coprolalia and copropraxia ) , the prevalence rates in ts patients have been observed to vary considerably,15,16 with rates of coprolalia reported in 21%,17 25%,18 33%,19 42%,20 and 43%;21 and copropraxia in 7%,18 17%,19 21%,17 and 23%21 of individuals with ts .
it is also to be noted that coprophenomena are not unique to tic disorders and their occurrence has been reported following stroke , fronto - temporal dementia , encephalitis , epilepsy , lesch nyhan syndrome , and choreoacanthocytosis.2224 in these instances of adult - onset tic disorders , there is often a potential trigger such as a neurological event with a temporal relationship to the onset of the symptoms , the symptoms are more severe with greater social morbidity , increased sensitivity , and poorer response to neuroleptic medication.24 a multi - site collaborative study undertaken by the tourette syndrome international database consortium involving a subset of 597 patients from 15 sites in seven countries , reported that coprolalia occurred at some time in 19% of males and 15% of females , and copropraxia in 6% of males and 5% of females.25 coprolalia was three times as frequent as copropraxia , with a mean onset of each at about 11 years , 5 years after the onset of tics . however , in 11% of those with coprolalia and 12% of copropraxia , these symptoms were one of the initial symptoms of ts.25 the most robust associations of coprophenomenon were with the number of non - tic repetitive behaviors , spitting and inappropriate sexual behavior .
this suggested that the course and impact are variable and that more research is required.25 given the varying results obtained in previous research assessing complex tics including the coprophenomenon , there is a need to better understand the prevalence and phenomenology of simple versus complex motor and vocal tics and associated phenomena .
furthermore , there is a need to evaluate the differences between individuals with pure - ts and ts - plus and simple versus complex tics .
a cohort of 400 patients attending specialized tourette clinics in the uk fulfilling diagnostic criteria for ts was recruited for the study .
a diagnosis of ts and any additional psychiatric diagnoses were made using either the dsm-111 to dsm - iv - tr26 edition of the diagnostic and statistical manual for mental disorders diagnostic criteria , depending on when the patients were assessed .
patients were clinically assessed and examined by the second author , an experienced neuropsychiatrist ( mmr ) using standardized schedules .
a subgroup of 222 ts patients who had full data on comorbidities and associated psychopathologies was selected for further analysis . within this sample ,
individuals with pure - ts ( motor and vocal tics only ) were compared with participants who reported associated comorbidities ( ts - plus ) .
each participant underwent a one - time detailed clinical interview using three published schedules for assessing individuals with ts including the national hospital interview schedule ( nhis ) for ts and related behaviours,27 the diagnostic confidence index ( dci),28 and the yale global tic severity scale ( ygtss).29 the nhis provides a standardized current diagnosis of ts characterized by multiple motor ( more than one ) and at least one vocal tic as per the diagnostic and statistical manual for mental disorders criteria .
the nhis was used as a structured method for assessment of tics as present at interview , in the previous week and ever ( lifetime ) based on self - report for adults and self- and parent - reports in the case of children and they were also evaluated for related features , relevant family history , and the presence of comorbid conditions including ocd , adhd , and any other co - existent psychopathology .
the dci provides a measure of whether an individual is likely to have met diagnostic criteria for ts in the past or currently .
the ygtss is a semi - structured clinician rated scale assessing tic severity based on the presence of tics in the previous week .
simple if it involved only a single group of muscles or a simple vocal / phonic sound.3 it is to be noted that invariably all patients who had complex tics also had simple tics .
data were analyzed using spss statistics version 22 ( ibm corporation , armonk , ny , usa ) , with alpha set at 0.05 for all comparisons .
relationships between levels of categorical data were examined using fisher s exact test , with phi values included to indicate the size of effects . for phi , values of 0.1 , 0.3 , and 0.5
independent - samples student s t - tests were conducted to explore group differences in continuous data , with cohen s d effect sizes also included .
cohen s d values of 0.2 , 0.5 , and 0.8 denote small , medium , and large effects respectively .
data were analyzed with the aim of examining the occurrences and inter - relationships between pure - ts ( motor and vocal tics only ) and ts - plus ( tics associated with other comorbid conditions ) and also between those with simple motor and vocal tics compared to those with complex motor and vocal tics including echophenomenon and complex vocalizations including coprophenomenon .
a cohort of 400 patients attending specialized tourette clinics in the uk fulfilling diagnostic criteria for ts was recruited for the study .
a diagnosis of ts and any additional psychiatric diagnoses were made using either the dsm-111 to dsm - iv - tr26 edition of the diagnostic and statistical manual for mental disorders diagnostic criteria , depending on when the patients were assessed .
patients were clinically assessed and examined by the second author , an experienced neuropsychiatrist ( mmr ) using standardized schedules .
a subgroup of 222 ts patients who had full data on comorbidities and associated psychopathologies was selected for further analysis . within this sample ,
individuals with pure - ts ( motor and vocal tics only ) were compared with participants who reported associated comorbidities ( ts - plus ) .
each participant underwent a one - time detailed clinical interview using three published schedules for assessing individuals with ts including the national hospital interview schedule ( nhis ) for ts and related behaviours,27 the diagnostic confidence index ( dci),28 and the yale global tic severity scale ( ygtss).29 the nhis provides a standardized current diagnosis of ts characterized by multiple motor ( more than one ) and at least one vocal tic as per the diagnostic and statistical manual for mental disorders criteria .
the nhis was used as a structured method for assessment of tics as present at interview , in the previous week and ever ( lifetime ) based on self - report for adults and self- and parent - reports in the case of children and they were also evaluated for related features , relevant family history , and the presence of comorbid conditions including ocd , adhd , and any other co - existent psychopathology .
the dci provides a measure of whether an individual is likely to have met diagnostic criteria for ts in the past or currently .
the ygtss is a semi - structured clinician rated scale assessing tic severity based on the presence of tics in the previous week .
simple if it involved only a single group of muscles or a simple vocal / phonic sound.3 it is to be noted that invariably all patients who had complex tics also had simple tics .
data were analyzed using spss statistics version 22 ( ibm corporation , armonk , ny , usa ) , with alpha set at 0.05 for all comparisons .
relationships between levels of categorical data were examined using fisher s exact test , with phi values included to indicate the size of effects . for phi , values of 0.1 , 0.3 , and 0.5
independent - samples student s t - tests were conducted to explore group differences in continuous data , with cohen s d effect sizes also included .
cohen s d values of 0.2 , 0.5 , and 0.8 denote small , medium , and large effects respectively .
data were analyzed with the aim of examining the occurrences and inter - relationships between pure - ts ( motor and vocal tics only ) and ts - plus ( tics associated with other comorbid conditions ) and also between those with simple motor and vocal tics compared to those with complex motor and vocal tics including echophenomenon and complex vocalizations including coprophenomenon .
of the 400 participants , 156 ( 39% ) had experienced coprolalia while 79 ( 20% ) reported having had copropraxia .
independent t - tests revealed a significant difference between participants experiencing coprolalia and those who were not on both the ygtss total score t(326)=5.9 , p=<0.0001 , cohen s d=0.66 ; and the dci total score t(214)=11.23 , p=<0.0001 , cohen s d=1.52 . in both instances ,
a significant relationship was also observed between the presence of coprolalia and the presence of copropraxia ( fisher s exact test , p=<0.0001 , =0.44 ) as well as echolalia ( fisher s exact test , p=<0.0001 , =0.35 ) with a higher proportion of participants with coprolalia also experiencing both copropraxia and echolalia .
there were no significant associations between coprophenomena and comorbidities . in the subset of 222 subjects where comorbidity data were available ,
13.5% reported tics in the absence of comorbid disorder ( pure - ts group ) and the remainder ( 86.5% ) reported various comorbidities ( ts - plus group ) .
independent t - tests revealed no significant differences between participants in the pure - ts group and ts - plus groups on either the ygtss total score t(165)=0.08 , p=0.93 , cohen s d=0.02 ; or the level of diagnostic confidence as indicated by the dci total score t(117)=1.70 , p=0.093 , cohen s d=0.50 .
it can be noted that results from the dci nonetheless approached significance , and the effect size in that instance was of medium size ( see table 2 ) .
the presence of coprolalia was higher ( 27.8% ) in those with ts - plus as compared to those with pure - ts ( 0% ) using the dci and the difference in proportions was statistically significant ( fisher s exact test , p=0.036 , =0.20 ) .
similarly the presence of copropraxia was higher ( 15.4% ) in the ts - plus group as compared to the pure - ts ( 6.3% ) group .
although this did not reach statistical significance due to low experimental power resulting from the small number of subjects , the relative risk of coprolalia in ts - plus relative to pure - ts was found to be 1.5 , with a relative risk of copropraxia of 2.4 .
further , individuals with coprolalia were found to have a significantly higher diagnostic confidence score t(115)=8.9 , p<0.0001 , cohen s d=2.0 ; and higher ygtss total tic severity score that approached statistical significance t(68)=1.74 , p=0.086 , cohen s d=0.47 .
thus there were significant correlations between the three measures utilized in the study using spearman s rho ( see table 3 ) .
no differences were found between the pure - ts and ts - plus groups in any of the other core features including echophenomenon ( echolalia and echopraxia ) , and/or palilalia , palipraxia , family history of tics , or comorbidity etc .
the only exception was that the pure - ts group was associated with no family history of ocd ( fisher s exact test , p<0.0001 , =0.34 ) .
analysis of those patients with only simple tics ( motor and vocal ) as compared to complex motor behaviors and vocalizations revealed that there were no significant differences in any of the core clinical features , or with regards to severity , or level of diagnostic confidence .
however , those with complex motor / vocal tics were significantly more likely to report a premonitory urge or sensation ( fisher s exact test , p=0.006 , =0.15 ) .
our finding that only 13.5% of ts patients have pure motor and vocal tics only ( ie , pure - ts ) is in keeping with previous international studies that have reported that around 10% of ts patients have only tics in both clinical25 and community30,31 settings .
the rate of coprolalia in our cohort was 39% which is higher than the previously reported 20% from an international database,25 but similar to previous cohorts from the same clinic which may be a reflection of the referral bias to this specialized ts clinic.20,21 our finding that the dci scores did not differ in those with pure - ts versus ts - plus suggests that the current diagnostic criteria are an accurate way of defining the core clinical syndrome of ts.13 further , we did not find any differences between individuals who exhibited simple motor or vocal tics as compared to those with complex movements and vocalizations .
we observed a significant association between complex motor and vocal tics and the presence of premonitory urges .
this may at least in part be due to the fact that patients may be more aware of complex tics ( motor and vocal ) when they occur and hence may have been more perceptive of the premonitory urges that precede these tics .
it may also be because of the activation of a particular neurophysiological component linked to a specific neuronal circuitry in the genesis of complex tics which also results in premonitory urges . in this regard , it has been observed that slightly different psychological factors drive self - reported premonitory urge ratings in patients with and without comorbidities32 and that different types of premonitory urges are associated with simple / complex tics and obsessive
compulsive symptoms.33 premonitory urges are an important symptom , occurring in about 90% of ts patients34,35 and are usually identified by patients after the age of 10 years.11 the relationship between the premonitory urges and tic severity is inconsistent , with some studies showing no association36,37 and others indicating a positive relationship.3841 it is plausible that the conflicting results observed in different studies may be a reflection of the proportion of subjects in the respective cohorts with complex motor / vocal tics as well as comorbidities .
our findings of an increased occurrence of coprophenomenon in ts - plus cases , coupled with an association found between coprolalia and higher ygtss and dci scores , is in keeping with a recent study that reported that coprophenomenon occurred when tics are most severe and was associated with comorbidity.18 thus it appears that , while there are no significant differences between the types of tics per se ( ie , simple or complex tics ) in terms of severity or associated clinical features , the presence of coprophenomenon may indicate a more severe form of the disorder and perhaps more extensive involvement of the cortico
, striatal and basal ganglia site of the circuitry may be involved in tics only , while the involvement of the frontal cortical end of the circuitry may result in more socially inappropriate behaviors.4 similarly , the presence of comorbidities may suggest additional circuitry being involved such as the basal ganglia
limbic circuits in ocd and so forth which in turn may point to the involvement of additional genetic or environmental factors.42 the finding in the current study that pure - ts was associated with no family history of ocd seems to suggest this .
it could be postulated that , in addition to specific ts gene(s ) that are involved in the development of motor and vocal tics in pure - ts , there may be additional genes ( eg , linked to ocd ) and/or environmental factors involved when ts is associated with other comorbidities and psychopathologies .
while there is little doubt that the vulnerability to developing tics is genetically determined , there are a number of other genetic , epigenetic , and environmental factors that contribute to the final clinical expression .
the multi - strike model43 suggests that the location and extent of involvement of the circuitry may also be determined by a multitude of factors . in this model ,
initial genetic vulnerability ( or the first strike ) results in specific structural and/or functional changes in key brain areas and predisposes the individual to develop tics and related symptoms .
the second strike causes disturbances in the neuronal circuitry and neurotransmitter levels in the brain regions that would normally support self - regulatory compensatory responses that serve to inhibit tic expression .
the third strike , that may occur through a number of other pre- and post - natal events , would exert its influence in determining the final clinical expression of ts including the severity and comorbidities.44 thus , it may be that different sites of the circuitry may be affected based on the different time points in the development ( intrauterine and postnatally ) when the second or third strike is occurring as different brain regions are formed during intrauterine development and then mature postnatally at different time points .
there would also be a number of other modulating factors including sex with the suggested sex - dependent difference in the phenotypic expression of the putative ts genes with males showing tic related behaviors and females exhibiting obsessive compulsive behaviours.45 future studies should therefore explore the genetic and phenotypic relationship in conjunction with environmental variables to account for the contribution of these factors in clinical presentation and severity to better understand the pathogenesis in pure - ts versus ts - plus . while the large sample size and detailed evaluation of patients using standardized instruments by experienced specialists with expertise in ts add to the strength of this study
, there are a number of limitations that would merit highlighting . in this regard , it is to be noted that this was a naturalistic cohort study of patients who attended a specialized ts clinic and that the assessor was not blind to the group status as to whether the participants belonged to the pure - ts versus ts - plus group .
further , the higher rates of coprophenomenon observed in this study may be a reflection of the referral bias to this tertiary clinic and this might limit the generalizability of our findings to other settings .
however , the finding that the majority of ts patients have associated comorbidities and psychopathologies suggests the need for comprehensive evaluation of ts patients .
further , it would appear that while those with pure - ts and having only simple motor and vocal tics may only need psycho - education , those with ts - plus and associated complex tics and coprophenomenon would require a combination of psychological and pharmacological intervention to address the tics as well as the associated psychopathologies .
thus , the detailed assessment of tics as well as the associated conditions has significant implications for the management of ts as early recognition of the associated comorbidities and co - existent psychopathologies can improve the overall outcome and quality of life in ts . | this study addressed several questions relating to the core features of tourette syndrome ( ts ) including in particular coprolalia ( involuntary utterance of obscene words ) and copropraxia ( involuntary and inappropriate rude gesturing ) . a cohort of 400 ts patients was investigated .
we observed that coprolalia occurred in 39% of the full cohort of 400 patients and copropraxia occurred in 20% of the cohort .
those with coprolalia had significantly higher yale global tic severity scale ( ygtss ) and diagnostic confidence index ( dci ) total scores and a significantly higher proportion also experienced copropraxia and echolalia .
a subgroup of 222 ts patients with full comorbidity data available were also compared based on whether they had pure - ts ( motor and vocal tics only ) or associated comorbidities and co - existent psychopathologies ( ts - plus ) .
pure - ts and ts - plus groups were compared across a number of characteristics including ts severity , associated clinical features , and family history . in this subgroup , 13.5% had pure - ts , while the remainder had comorbidities and psychopathologies consistent with ts - plus .
thirty - nine percent of the ts - plus group displayed coprolalia , compared to ( 0% ) of the pure - ts group and the difference in proportions was statistically significant .
the only other significant difference found between the two groups was that pure - ts was associated with no family history of obsessive compulsive disorder which is an interesting finding that may suggest that additional genes or environmental factors may be at play when ts is associated with comorbidities .
finally , differences between individuals with simple versus complex vocal / motor tics were evaluated .
results indicated that individuals with complex motor / vocal tics were significantly more likely to report premonitory urges / sensations than individuals with simple tics and ts .
the implications of these findings for the assessment and understanding of ts are discussed . |
tuberculosis ( tb ) remains a worldwide health problem causing morbidity and mortality particularly in the developing and low - income countries .
there are several reports of tb occurring during or following the treatment of malignant hemopathy and stem cell transplantation [ 1 , 2 , 3 ] .
tb , especially the extrapulmonary subtype , is reputed as one of the great mimickers of malignancy .
we report a case of tb meningitis and lymphadenitis mimicking relapse of burkitt 's lymphoma .
a 53-year - old indian male known to have diabetes mellitus type 2 and hypertension was diagnosed at the end of 2009 with epstein - barr virus ( ebv)-negative , stage ivb burkitt 's lymphoma with liver involvement .
he was treated with a rituximab plus codox - m / ivac regimen ( codox - m : cyclophosphamide 800 mg / m on days 1 + 2 , vincristine 1.4 mg / m on days 1 + 8 , doxorubicin 50 mg / m on day 1 , methotrexate 3,000 mg / m on day 10 and rituximab 375 mg / m on day 8 ; ivac : ifosfamide 1,500 mg / m on days 15 , etoposide 60 mg / m on days 15 , cytarabin 2,000 mg / m on days 1 + 2 and rituximab 375 mg / m on day 4 ) . he received four cycles and 6 treatments of intrathecal methotrexate 12 mg .
the therapy was completed in march 2010 , and he was in complete remission as evaluated using a computed tomography ( ct ) scan . during the routine follow - up at 15 months posttreatment , he was in a good general condition but complained of left hip pain .
the ct scan of the whole body ( july 19 , 2011 ) revealed multiple subcentimetric lymph nodes in the mediastinal , retroperitoneal and para - aortic regions , and also showed the presence of an osteolytic lesion with a sclerotic margin at the left femoral head .
the magnetic resonance imaging scan of the femoral head showed a geographical benign looking and slowly growing focal osseous lesion at the proximal left femoral metaphysis .
subsequently , a follow - up f - fludeoxyglucose - positron emission tomography ( fdg - pet)/ct scan in june 2012 ( figure 1a , d ) showed intense tracer uptake [ standardized uptake value ( suv ) 32.4 ] in several 8- to 18-mm - sized lymph nodes at the thoracic inlet , mediastinum ( paravascular , paratracheal , subcarinal and retrocrural regions ) and in the abdomen ( para - aortic and paracaval regions ) .
the known sclerotic lesion in the left femoral head showed no increased fdg uptake favoring benign origin .
fever , night sweats , weight loss , palpable peripheral lymph node , hepatosplenomegaly and skin lesions were absent .
the decision was to rule out infection / connective tissue disease and to repeat imaging after 34 months because the lymph nodes were small and not accessible for ct - guided biopsy or surgery .
his laboratory investigations including viral serology study for herpes simplex type 1 , type 2 , ebv , cytomegalovirus ( cmv ) , human immunodeficiency virus , hepatitis b , hepatitis c and toxoplasmosis were negative .
complement ( c3 and c4 ) tests and immunoglobulin levels ( igg , iga and igm ) were normal . a repeat f - fdg - pet / ct scan in october 2012 ( figure 1b , e ) revealed an increase in the size of the previously reported lymph nodes and newly developed active lymph nodes on both sides of the diaphragm .
one week prior to the biopsy appointment , he presented to the emergency department with headache , vomiting and photophobia . on physical examination ,
he had a low - grade fever ( 37.8c ) and stiffness of the neck , but he was conscious , oriented and had no neurological deficit or cranial nerve involvement .
the complete blood count revealed the following : white blood cell ( wbc ) 8.4 10/l , neutrophil count 70% , lymphocyte count 30% , hemoglobin 14 g / dl , platelets 170 10/l .
glucose ( 9.8 mmol / l ) , electrolytes , renal function , liver function , lactate dehydrogenase and coagulation workup were normal .
the csf test revealed the following : wbc 622 10/l , with 84% lymphocytes , 6% monocytes and 10% neutrophils , glucose 2 mmol / l and protein 2.5
he was started on intravenous ceftriaxone and the next day was transferred to the hematology floor for suspicion of relapse of burkitt 's lymphoma of the central nervous system .
further investigations included magnetic resonance imaging of the brain , which showed mild diffuse meningeal enhancement .
lp was repeated , and intrathecal treatment with 15 mg methotrexate and 50 mg hydrocortisone was given .
the wbc count was 908 10/l , with 96% lymphocytes and 4% neutrophils , glucose 2.5 mmol / l and protein 3.5
gram stains and culture were negative ; acid - fast bacilli smear and tb pcr were negative .
the csf pcr for cmv , ebv , adenovirus , herpes simplex type 1 and 2 , varicella virus , enterovirus and mumps virus was negative .
csf findings of low glucose concentration , elevated protein and polyclonal lymphocytosis were more suggestive of tb meningitis than lymphoma ; hence , he was started empirically on antituberculosis ( a combination of four drugs : isoniazid , rifampicin , pyrazinamide and ethambutol ) and steroid treatment .
a third lp after 48 h of anti - tb showed a decrease in wbc count ( 182 10/l , with 88% lymphocytes , 11% neutrophils and 1% monocytes ) . bacterial gram stain and culture were negative ; acid - fast bacilli smear and pcr were negative .
a ct scan on december 5 , 2012 , showed absence of pleuropulmonary lesions and the same findings as in the previous imaging .
bone marrow biopsy was normal and showed absence of granuloma , and tb culture on bone marrow was negative .
csf tb culture and tb pcr ( genexpert ) were positive , confirming the diagnosis of mycobacterium tuberculosis , which is sensitive to conventional anti - tb .
the diagnosis of tb meningitis and lymphadenitis was established , and the patient continued taking the antituberculosis drugs for 2 months followed by isoniazid and rifampicin for 10 more months . repeat f - fdg - pet / ct at the end of treatment revealed a total regression of lymph nodes ( figure 1c , f ) .
tb infection is a serious and life - threatening complication in patients with malignant hemopathy and in recipients of bone marrow transplant , especially in endemic areas .
the increased risk of reactivation of latent tb could be explained by severely impaired cell - mediated immunity as a result of the underlining disease and its immunosuppressive treatment [ 1 , 2 , 3 ] .
the real incidence of tb in hematological malignancies and stem cell transplantation is not well identified ; radiological and epidemiological data are based on case reports and retrospective analyses with small cohorts of patients . in taiwan , tb occurs in approximately 120 per 100,000 adult patients with hematological malignancy .
extrapulmonary disease is common in patients with hematological disorders , ranging from 16 to 70% for all kinds of tb disease [ 3 , 4 ] .
tb can precede or be concomitant with the diagnosis of the malignancy or occur following treatment of malignant disorders [ 1 , 3 , 4 ] .
the average time between the completion of cytotoxic chemotherapy and the development of tb is 1820 months .
in addition , the diagnosis of tb is often difficult to determine due to its atypical clinical presentation and radiological findings .
tb meningitis accounts for 1% of all tb cases and for 6% of all extra pulmonary infections in immunocompetent patients .
it is a life - threatening complication ; the morality rate remains high at 1540% despite effective treatment .
in immunocompromised patients with malignant hemopathy , only few sporadic cases have been reported [ 7 , 8 ] . in the present case ,
the occurrence of mediastinal and retroperitoneal hypermetabolic lymph nodes which gradually increased in size over 1 year followed by lymphocytic meningitis were highly suggestive and mistaken for a recurrence of burkitt 's lymphoma ; however , the slow progression of the disorder and the small size of the lymph node was inconsistent with relapse of burkitt 's lymphoma , which is a highly proliferative disease .
the delay in the diagnosis of tb lymphadenitis after the first pet / ct scan was due to the small size of the lymph node that did not allow biopsy , the negative tuberculin skin test and quantiferon , and absence of pleuropulmonary lesion on the consecutive imaging .
the tuberculin skin test is usually negative in patients with malignant hemopathy because of the defect in the immunity related to the disease itself and to cytotoxic chemotherapy [ 1 , 3 , 4 ] .
the quantiferon tb gold in - tube test reflects the release of interferon- and is highly recommended in the screening of latent tb in patients with malignant hemopathy and stem cell transplant ; its sensitivity and specificity are around 75 and 81% , respectively .
both tests were negative before and after confirmation of tb in our patient , reflecting the severe defect of his immunity .
f - fdg - pet imaging has a well - documented lack of specificity in discriminating infection and inflammation from malignancy mainly due to the increased macrophage and neutrophil tracer uptake [ 10 , 11 , 12 ] .
differentiation between fdg - avid malignancies such as burkitt 's lymphoma and active tb based on suvmax is not possible since they both demonstrate intense hypermetabolism with an overlap in suv values when quantified .
furthermore , the dual time point imaging could not differentiate malignant lymph node involvement from tuberculous lymphadenitis either .
for this reason , the f - fdg - pet / ct imaging characteristics of tb can be easily confused with malignancy , and therefore positive f - fdg - pet / ct results should be interpreted with caution when differentiating benign from malignant disease .
the distribution of the f - fdg - avid foci in correlation with the clinical signs could suggest tb infection ; however , in the case of a nonsymptomatic lymphadenitis and absence of pleuropulmonary lesions in a cancer patient who underwent immunosuppressive treatment , there are no definite investigational modalities to confirm the diagnosis other than histopathological examination and tb culture . in spite of its low specificity to diagnose tb , f - fdg - pet /
to detect active tuberculous lesions allows correct evaluation of the extrapulmonary disease extent , which might have an impact on the treatment plan .
treatment response assessment appears to be the most promising application of f - fdg - pet / ct in tb .
it allows early evaluation of treatment response and differentiates between responders and nonresponders at 2 months and at the end of treatment [ 15 , 16 , 17 ] .
early evaluation of treatment response is essential in the management of patients with tb , particularly extrapulmonary tb , when multidrug resistance is expected and treatment modification is necessary in nonresponders at an early stage of the disease [ 18 , 19 ] .
results suggest that f - fdg - pet / ct may be useful in previously healed tb cases , as patients with pulmonary sequelae demonstrating higher suv ( 1.5 ) on f - fdg - pet / ct likely have a higher risk for subsequent tb activation .
we have highlighted the fact that the increased fdg uptake might not necessarily be an indication of high - grade lymphoma relapse , and tb should be checked for especially in patients from endemic areas .
the presented case also exemplifies the potential of f - fdg - pet / ct imaging in early detection and assessment of the extent of extrapulmonary tb in immunosuppressed cancer patients .
| tuberculosis ( tb ) can present with various forms and can occasionally be mistaken for malignancy .
hereby , we report a 53-year - old man diagnosed and treated for burkitt 's lymphoma in 2009 who achieved a complete remission confirmed by a computed tomography ( ct ) scan . during the follow - up 2 years later , he complained of left hip pain that warranted investigation with magnetic resonance imaging and whole - body 18f - fludeoxyglucose - positron emission tomography ( fdg - pet)/ct which showed a benign lesion in the left hip associated with multiple lymph nodes in the chest and abdomen not amenable for biopsy . a follow - up
pet / ct scan a few months later showed intense tracer uptake in the lymph nodes with size progression and appearance of new lymph nodes suspicious of lymphoma relapse .
the patient was asymptomatic , and all investigations including viral and connective tissue disease studies were negative . also the tuberculin skin test and quantiferon were negative .
lymph node biopsy was planned ; however , the patient presented a few days earlier with fever , headache and photophobia .
cerebrospinal fluid ( csf ) examination confirmed meningitis with lymphocytic pleocytosis and elevated protein . the csf gram stain , culture , viral and acid - fast bacilli were negative .
csf flow cytometry and cytopathology confirmed polyclonal lymphocytosis and suggested reactive causes .
csf tb culture grew mycobacterium tuberculosis .
mediastinal lymph node biopsy also confirmed tb lymphadenitis .
four antituberculosis drugs were started .
one year later , a pet / ct scan showed regression of all the involved lymph nodes .
this case highlights the importance of excluding tb in patients with suspected malignancy , especially if they belong to endemic regions , and the increasing role of 18f - fdg - pet / ct in the early detection of extrapulmonary tb . |
the accelerated global industrialisation trends during the past century have raised many concerns about the potential environmental health problems that might be caused as a result . in 2012
, it was estimated that seven million global deaths per year were attributed to air pollution.1 enhanced by the lack of strict environmental controls and public health systems in developing countries , industrialisation has shifted in recent decades from developed to developing countries , deteriorating environmental and public health situations in the latter.2 a recent report of the who shows that 25% of the mortality in developing countries is environmentally related ; this estimate is 17% in developed countries .
children are particularly susceptible to environmental exposures,3 and the who 's recent estimates show that 36% of the global child mortality was attributable to environmental causes.4 oman , one of the currently fast developing economies , has also initiated a heavy industrialisation movement to supplement its national resources , leading to a rapid establishment of several major industrial parks in the country .
one of these is the sohar industrial zone ( siz ) , a large petrochemical and heavy metal industrial complex .
the establishment of this complex near a densely populated residential area consequently raised great health , environmental and financial concerns from its surrounding residents .
siz , with its anticipated expansions in the near future , necessitates a parallel community health assessment of the population of the residential area around it .
evidence elsewhere shows that children living near industrial establishments have a higher risk of adverse health outcomes when compared with those living away.510 however , global evidence on the risk of adverse health of young populations living in proximity to industries is still limited , as highlighted in a recent review by pascal et al.11 a recent study , also the first environmental epidemiological study in oman , showed increased risk of respiratory and allergic disease in an adult population living in proximity to siz.12 no similar study has been carried out in oman to assess the link between the recent industrial developments and child health .
the aim of this study was to examine the relationship between adverse health and proximity to siz in a young population .
daily patient visits to state health clinics in the two provinces around siz , sohar and liwa , were obtained from the national al - shifa electronic system , omani ministry of health ( moh ) for the period of 1 january 2006 to 31 december 2010 .
information for each patient visit included the consultation date , a unique patient identification number , age , village , health centre and the diagnosis based on the international classification of diseases 10th revision ( icd-10 ) .
ethical approval for this study was obtained from the moh and brunel university ethical committee . according to previous evidence on the health effects of air pollution in young populations
respiratory diseases included : acute respiratory diseases ( ard ) defined as upper ( icd-10 : j0-j06 ) , other acute lower respiratory infections ( icd-10 : j20-j22 ) and pneumonia ( icd-10 : j12-j18 ) ; and asthma ( icd-10 : j45 and j46 ) .
allergic diseases included disorders of the conjunctiva ( icd-10 : h10-h13 ) , dermatitis ( icd-10 : l20-l30 ) and eczema ( icd-10 : l50-l54 ) .
area - specific population by age and gender for each village was obtained from the omani national census of 2010 , and was used as an offset in the model .
the education and occupational status of the residents was also obtained from the same source and utilised to define socioeconomic status ( ses ) . daily temperature and
wind data were provided by the omani department of meteorology for the entire study period . for this study , it was important to distinguish between visits for a
new event and a follow - up for the selected diseases . in previous epidemiological studies , definitions of a
new case of ard in children vary from 1 week disease - free duration to up to 3 months.13
14 for asthma , many epidemiological studies define a new asthma attack in children as an attack happening after one attack - free year.15 other studies have used periods of 1 day,16 2 weeks,17 up to 1 month attack - free for their definitions.18 for allergic diseases , several epidemiological studies extended their definition of a new event from 10 disease - free days19 to up to 1 year.20 from the above discussion , and also to ensure sufficient sample size for the analysis , we used a 1 month period to define
new events. siz is composed of the sohar industrial port ( sip ) and the sohar industrial estate ( sie ) .
the petrochemical complex started to operate with an oil refinery in 2006 , and expanded in the following 3 years to include the polypropylene , methanol , formaldehyde , urea and aromatics industries .
the main industry in sie is an aluminium smelter , which started to operate in 2008 .
owing to its early operation date and subjectively visible plumes , we considered the oil refinery ( in sip ) as the centre point of the industrial park used in the exposure assessment method .
air pollution monitoring and measurements in the area were not available for the study period .
we employed a proximity method to classify exposure of the study population according to their distance from the source of the industrial pollution .
the proximity method has been used frequently in many influential environmental epidemiology studies with considerable impact on public health practices and policies.5
2123 the method is also used by many environmental regulatory bodies to outline environmental policies , for example , the us environmental protection agency environmental exposure risk assessment framework,24 and the uk health and safety excusive in their planning advice for land use near a major hazard.25 the method has also been used in the quick assessments of industrial accidents,26 and in environmental justice studies which often apply this method in their assessment of environmental inequality.27 to assist us in determining the exposure categories , we reviewed previous relevant epidemiological and policy studies . in some of these studies , the decision of the minimum distance for the proximity method was arbitrary,28
29 using the residents complaints of pollutant odours22 or determined by environmental measurements.30 definitions of exposure categories in these studies varied from a 5 to 20 km radius from the industries of concern.11
22
31 additionally , we utilised several examples of international housing policies , which recommend the minimal safe distance of housings from industrial areas , generally at > 2 km.32 taking into consideration this previous evidence and to increase the power of the study , we decided on an incremental distance of 5 km from the refinery to classify the exposure area around siz .
three exposure zones were defined : high , for those living 5 km from the refinery ; intermediate , those living within > 5 to 10 km from the refinery ; and control zone as living 20 km from the refinery .
there were no villages located between 10 and 20 km from the refinery ; therefore , this distance was not represented in the study ( figure 1 ) .
the use of only state health data resulted in a small number of cases from sohar city , the main metropolitan area of the study provinces . in its latest statistics ,
moh has registered 51 private health clinics in the study area , out of which 94% were located in sohar city . therefore , the city was excluded from the analysis .
sip , sohar industrial port ; sie , sohar industrial estate . to ensure validity of our proximity exposure definitions ,
the prevailing winds pointed to the west during summer and september , suggestively carrying the pollutants farther inland .
wind roses pointed towards the seaside in winter and november , and had no major direction in the remaining months .
this indicated that during summer and september , the high and intermediate exposure zones could most likely reveal similar pollutant patterns .
we also tested a dispersion model of so2 emitted from the oil refinery for a very limited period of emission data on pollutant concentrations ( between 1 and 15 january 2008 ) , and other emission parameters including stack parameters such as number of stacks , location , height and diameter , emission rate , temperature and velocity .
the model suggested a relative change of two to four times in the predicted so2 concentrations for distances 510 km , supporting our proximity exposure classification .
modelling diagnostics ( akaike 's information criterion ) suggested the use of a negative binomial distribution to control for the overdispersion in the data .
time , as a month number , was defined as a continuous variable , and smoothing splines were applied to capture the non - linear seasonal and longer term trends in the selected diseases .
we defined three age groups for the analyses : infants as 1 year old , children as > 1 to 14 and young adults as > 14 to < 20 .
exposure zones ( with control zone as the reference ) were coded as factors in the models .
the logarithms of gender and age group - specific population data were used as an offset in the model .
mean daily temperature , season and individual years were tested as potential confounders , and were not found to affect the exposure health relationship .
this lack of effect could partially be due to the control for time by smoothing function in the model .
risk ratio ( rr ) for the different exposure zones was calculated as exp and the 95% ci levels were calculated using the formula : exp .
owing to the similarities of the rr between the high and intermediate exposure zones , further models were carried out combining the two exposure zones .
this was also supported by the similarity of the likely pollutant spread between the two exposure zones , as suggested by the wind roses and dispersion model . to further explore the relationship between exposure and the studied diseases , monthly follow - up frequencies for ard and asthma
were also modelled , using the same model structure and stratified according to age and gender groups .
ses for each village was determined as the proportions of the total adult population with no education ,
high education and employment , which are representatives of the parents education and employments .
the distributions of each ses index , across all 59 villages , were divided into two strata : below and above 50% .
ses stratified analyses were only carried out for ard , due to insufficient numbers for other disease definitions .
daily patient visits to state health clinics in the two provinces around siz , sohar and liwa , were obtained from the national al - shifa electronic system , omani ministry of health ( moh ) for the period of 1 january 2006 to 31 december 2010 .
information for each patient visit included the consultation date , a unique patient identification number , age , village , health centre and the diagnosis based on the international classification of diseases 10th revision ( icd-10 ) .
ethical approval for this study was obtained from the moh and brunel university ethical committee . according to previous evidence on the health effects of air pollution in young populations
respiratory diseases included : acute respiratory diseases ( ard ) defined as upper ( icd-10 : j0-j06 ) , other acute lower respiratory infections ( icd-10 : j20-j22 ) and pneumonia ( icd-10 : j12-j18 ) ; and asthma ( icd-10 : j45 and j46 ) .
allergic diseases included disorders of the conjunctiva ( icd-10 : h10-h13 ) , dermatitis ( icd-10 : l20-l30 ) and eczema ( icd-10 : l50-l54 ) .
area - specific population by age and gender for each village was obtained from the omani national census of 2010 , and was used as an offset in the model .
the education and occupational status of the residents was also obtained from the same source and utilised to define socioeconomic status ( ses ) . daily temperature and
wind data were provided by the omani department of meteorology for the entire study period . for this study , it was important to distinguish between visits for a
new event and a follow - up for the selected diseases . in previous epidemiological studies , definitions of a
new case of ard in children vary from 1 week disease - free duration to up to 3 months.13
14 for asthma , many epidemiological studies define a new asthma attack in children as an attack happening after one attack - free year.15 other studies have used periods of 1 day,16 2 weeks,17 up to 1 month attack - free for their definitions.18 for allergic diseases , several epidemiological studies extended their definition of a new event from 10 disease - free days19 to up to 1 year.20 from the above discussion , and also to ensure sufficient sample size for the analysis , we used a 1 month period to define
for this study , it was important to distinguish between visits for a new event and a follow - up for the selected diseases . in previous epidemiological studies , definitions of a new case of ard in children vary from 1 week disease - free duration to up to 3 months.13
14 for asthma , many epidemiological studies define a new asthma attack in children as an attack happening after one attack - free year.15 other studies have used periods of 1 day,16 2 weeks,17 up to 1 month attack - free for their definitions.18 for allergic diseases , several epidemiological studies extended their definition of a
new event from 10 disease - free days19 to up to 1 year.20 from the above discussion , and also to ensure sufficient sample size for the analysis , we used a 1 month period to define
siz is composed of the sohar industrial port ( sip ) and the sohar industrial estate ( sie ) .
the petrochemical complex started to operate with an oil refinery in 2006 , and expanded in the following 3 years to include the polypropylene , methanol , formaldehyde , urea and aromatics industries .
the main industry in sie is an aluminium smelter , which started to operate in 2008 . owing to its early operation date and subjectively visible plumes , we considered the oil refinery ( in sip ) as the centre point of the industrial park used in the exposure assessment method .
air pollution monitoring and measurements in the area were not available for the study period .
we employed a proximity method to classify exposure of the study population according to their distance from the source of the industrial pollution .
the proximity method has been used frequently in many influential environmental epidemiology studies with considerable impact on public health practices and policies.5
2123 the method is also used by many environmental regulatory bodies to outline environmental policies , for example , the us environmental protection agency environmental exposure risk assessment framework,24 and the uk health and safety excusive in their planning advice for land use near a major hazard.25 the method has also been used in the quick assessments of industrial accidents,26 and in environmental justice studies which often apply this method in their assessment of environmental inequality.27 to assist us in determining the exposure categories , we reviewed previous relevant epidemiological and policy studies . in some of these studies ,
the decision of the minimum distance for the proximity method was arbitrary,28
29 using the residents complaints of pollutant odours22 or determined by environmental measurements.30 definitions of exposure categories in these studies varied from a 5 to 20 km radius from the industries of concern.11
22
31 additionally , we utilised several examples of international housing policies , which recommend the minimal safe distance of housings from industrial areas , generally at > 2 km.32 taking into consideration this previous evidence and to increase the power of the study ,
we decided on an incremental distance of 5 km from the refinery to classify the exposure area around siz . three exposure zones were defined : high , for those living 5 km from the refinery ; intermediate , those living within > 5 to 10 km from the refinery ; and control zone as living 20 km from the refinery .
there were no villages located between 10 and 20 km from the refinery ; therefore , this distance was not represented in the study ( figure 1 ) .
the use of only state health data resulted in a small number of cases from sohar city , the main metropolitan area of the study provinces . in its latest statistics ,
moh has registered 51 private health clinics in the study area , out of which 94% were located in sohar city . therefore , the city was excluded from the analysis .
sip , sohar industrial port ; sie , sohar industrial estate . to ensure validity of our proximity exposure definitions , we constructed monthly wind roses for the study period using wind speed and direction .
the prevailing winds pointed to the west during summer and september , suggestively carrying the pollutants farther inland .
wind roses pointed towards the seaside in winter and november , and had no major direction in the remaining months .
this indicated that during summer and september , the high and intermediate exposure zones could most likely reveal similar pollutant patterns .
we also tested a dispersion model of so2 emitted from the oil refinery for a very limited period of emission data on pollutant concentrations ( between 1 and 15 january 2008 ) , and other emission parameters including stack parameters such as number of stacks , location , height and diameter , emission rate , temperature and velocity .
the model suggested a relative change of two to four times in the predicted so2 concentrations for distances 510 km , supporting our proximity exposure classification .
monthly event counts were modelled using a generalised additive model . modelling diagnostics ( akaike 's information criterion ) suggested the use of a negative binomial distribution to control for the overdispersion in the data .
time , as a month number , was defined as a continuous variable , and smoothing splines were applied to capture the non - linear seasonal and longer term trends in the selected diseases .
we defined three age groups for the analyses : infants as 1 year old , children as > 1 to 14 and young adults as > 14 to < 20 .
exposure zones ( with control zone as the reference ) were coded as factors in the models .
the logarithms of gender and age group - specific population data were used as an offset in the model .
mean daily temperature , season and individual years were tested as potential confounders , and were not found to affect the exposure health relationship .
this lack of effect could partially be due to the control for time by smoothing function in the model .
risk ratio ( rr ) for the different exposure zones was calculated as exp and the 95% ci levels were calculated using the formula : exp .
owing to the similarities of the rr between the high and intermediate exposure zones , further models were carried out combining the two exposure zones .
this was also supported by the similarity of the likely pollutant spread between the two exposure zones , as suggested by the wind roses and dispersion model . to further explore the relationship between exposure and the studied diseases , monthly follow - up frequencies for ard and asthma
were also modelled , using the same model structure and stratified according to age and gender groups .
ses for each village was determined as the proportions of the total adult population with
no education , high education and employment , which are representatives of the parents education and employments .
the distributions of each ses index , across all 59 villages , were divided into two strata : below and above 50% .
ses stratified analyses were only carried out for ard , due to insufficient numbers for other disease definitions .
to further explore the relationship between exposure and the studied diseases , monthly follow - up frequencies for ard and asthma were also modelled , using the same model structure and stratified according to age and gender groups .
ses for each village was determined as the proportions of the total adult population with
no education , high education and employment , which are representatives of the parents education and employments .
the distributions of each ses index , across all 59 villages , were divided into two strata : below and above 50% .
ses stratified analyses were only carried out for ard , due to insufficient numbers for other disease definitions .
the total population - at - risk of census 2010 was 25 215 ( table 1 )
. a greater proportion of males in the study population was noted in particular among the ages > 1 to < 20 years , and it was more pronounced in the high exposure group .
a similar pattern of unequal gender distribution was observed in the population - at - risk .
characteristics of the study population for each exposure zone population ages < 20 years , sohar and liwa provinces , oman , 20062010 . *
percentage to the total counts in the exposure zone . showing the mean percentage of the respective ses in the villages of each exposure zone with the sd .
the total number of villages in the study is 59 ; 8 are located in the high exposure zone ; 4 are located in the intermediate zone ; the remaining 47 smaller villages are located in the control zone .
ard , acute respiratory diseases ; f , female ; m , male ; ses , socioeconomic status . the results of the model analysis for the association of the number of new visits for the selected diseases and exposures are presented in table 2 .
high and intermediate exposure zones had greater effects for all diseases when compared with the control exposure zone .
the consistency in the similarity of the associations for the two exposure zones suggested the feasibility of combining them into one zone of 10 km radius from the oil refinery .
multivariable analysis * of acute respiratory diseases , asthma , conjunctivitis and dermatitis incidence , in association with the high and intermediate exposure zones study population of ages < 20 years , sohar and liwa provinces , oman , 20062010 . * adjusted for time trend .
table 3 shows the results of the analysis combining the high and intermediate exposure zones stratified by age category , gender and ses for ard .
positive associations for all diseases in the combined exposure zone were observed , compared with the control exposure zone .
the findings suggested no gender difference in exposure effects and no difference by age for ard , conjunctivitis and dermatitis .
multivariable analysis * of acute respiratory diseases , asthma , conjunctivitis and dermatitis incidence , in association with the combined exposure zone study population of ages < 20 years , sohar and liwa provinces , oman , 20062010 . * adjusted for time trend .
stratification by ses was only carried out for ard due to insufficient numbers in other disease definitions .
ard , acute respiratory diseases ci , confidence interval ; rr , risk ratio ; ses , socioeconomic status . table 4 shows the results of the model analysis for the follow - up frequencies of ard and asthma .
increased follow - up frequencies were observed in the combined exposure zone when compared with the control .
multivariable analysis * of acute respiratory disease and asthma follow - ups in association with the combined exposure zone study population of ages < 20 years , sohar and liwa provinces , oman , 20062010 .
follow - ups were defined as any repeated patients visits within 1 month of two new event visits .
this study was carried out to investigate the relationship between adverse health effects in a young population living in proximity to a new industrial park in oman .
the findings of the study suggested an increase of more than twofold to threefold in the risk of respiratory and allergic diseases among a young population living within 10 km of the industrial park .
this geographical dispersion of pollution could be explained by the unfavourable geographic location of siz .
a recent meteorological study by al - khadouri et al33 reported that the location of siz promotes the formation of a stagnant air mass that could travel more than 10 km inland .
owing to the lack of pollutant emission data for this study , we used the proximity to source for our exposure classification .
this approach has been used frequently in many environmental epidemiological,5
2123 environmental policies,24
25 and environmental justice studies.27 this extensive use of the proximity method as an exploratory tool for scoping health effects around industrial areas is mainly because it is simple , fast and more economical , and in our study it was the only way to assess the health effects of this rapidly developing industrial setting .
our results agree with the existing epidemiological evidence regarding the health effects of the respiratory tract system,5 eye6 and skin7 of children living near a petrochemical complex .
these industries are known to emit a number of environmental pollutants including so2 , nitric oxides ( nox ) and volatile organic compounds,34 which have been shown to cause skin and mucosal lining irritation , especially of the eyes and respiratory tract.35 several epidemiological studies reveal that children living in the proximity of aluminium and iron smelters are prone to impaired lung function,9 increased hospitalisation from respiratory disease,8 and increased allergic sensitisation and allergic symptoms,10 most likely related to emitted aluminium and fluoride compounds , so2 , nox and heavy metals.34 the findings of this study did not show any difference in exposure disease associations by age groups ; an indication of a greater association between asthmatic events and exposure among ages 14 years was observed .
this is most likely due to the susceptibility of these age groups because of less mature respiratory and immune systems , and also because children spend most of their time outdoors and breathe 50% more air per body mass compared with adults.3 a greater number of males aged > 1 to < 20 years was noted in the exposure zones for both study and at - risk population groups .
gender differences were not as pronounced in the control zone . to understand these patterns , we analysed the omani census data of 2010 to compare the study population structure with the entire omani population .
the results suggested that the difference in the gender ratio in the study area starts from ages 10 to 14 years , a phenomenon not observed in the entire omani population .
one possible explanation for this greater number of young males in the study area might be due to the increasing labour force migration to this industrial area .
this was also supported by the world bank statistics of 2009 , which showed that 87% of the young ( aged 1524 years ) labour force in oman was males.36 this is the first epidemiological study carried out in oman that assessed the health of children living in proximity of an industrial complex .
similar studies elsewhere confirm our findings of adverse health effects among the young population and children living in the proximity of industrial settings.510 this evidence is , however , limited as highlighted by a recent review by pascal et al11 which identified only 17 such studies globally .
therefore , we believe that the contribution of this study is important from a country , regional and global perspective . the findings of this study , in corroboration with the limited previous evidence , are particularly important from the perspective of the shift in environmental burden of disease from developed to developing countries .
the accelerated need for economic development in many developing countries has led to the movement of the industrialisation trend from developed to developing countries .
this has negatively affected the state of environment and health in these countries , particularly due to the lack of strict environmental and public health policies.2 in oman , this inevitable industrial development has potential disadvantaged environmental situations , which may also contribute to further adverse health in population .
the rapid pace of this industrial development trend requires the establishment and strengthening of environmental and public health systems in the country .
we believe that the use of a precautionary principle to protect public and community health around any new industrial parks in oman would be an appropriate early step to protect population health by the great environmental and health risks posed by this rapid industrial development.37 as a preventive measure , the omani government has planned to displace the population in proximity of the siz industrial park to a different residential area .
this measure , involving large economic costs,38 has potential sociocultural and economic implications for the displaced population , which merit further consideration .
future intervention programmes that focus on health protection need to consider these sociocultural factors , in addition to proper planning for environmental monitoring and control
. a few limitations of this study need to be recognised . owing to the absence of emission data
the use of this approach might introduce misclassification bias when assigning exposure to population groups as pollutants do not respect boundaries.39 however , we tried to minimise this bias by assessing the potential extent of the pollutant spread utilising wind roses and dispersion model .
these distances were also confirmed by similar definitions in previous epidemiological and policy studies.5
31 another source of bias was potentially due to the study population loss to private healthcare .
however , we think that this source of bias is small in our study , after excluding sohar city from the analyses . there is great provision and uniform distribution of state health centres ( large and medium ) in exposure and control zones , which ensure easy access to free healthcare , and only a very limited number of small private clinics ( only 3 from a total of 51 private clinics in the larger district).40 also , excluding sohar city from the analyses
the use of morbidity healthcare visit data could introduce inaccuracy in defining health outcomes , due to physician inaccuracies in diagnosis , patient comorbidities and the parents threshold to seek medical help for their children.41 the latter also depends on sociocultural factors and on accessibility to healthcare . while other factors are more difficult to control in an epidemiological study , the uniformly distributed state health institutions in the study area ensured easy accessibility and most likely uniform reporting and diagnosis of the health conditions studied here .
this study is the first environmental health study conducted in oman to investigate health effects in children living near an industrial complex . this study would hope to mark the start of subsequent environment and health research and monitoring , and to assist in building up evidence for public health practice and environmental health policy in oman .
we also hope that these findings will contribute to a triggering of preventive actions and public health intervention programmes , as well as to global evidence on the effects of industrial development and child health .
what is already known on this subjectliving in proximity to industrial establishments is associated with adverse health in populations .
what this study addsthis study is the first environmental epidemiological study in oman that assessed the link between proximity to an industrial park and health in a young population.the study adds to the currently limited global evidence on the link between proximity to industry and child health .
this study is the first environmental epidemiological study in oman that assessed the link between proximity to an industrial park and health in a young population .
the study adds to the currently limited global evidence on the link between proximity to industry and child health . | backgroundsohar industrial zone ( siz ) , oman , which started operating in 2006 , contains many industries that potentially affect the health of the local population .
this study 's aim was to evaluate the health effects in a young population living near siz.methodspatient visits to state health clinics for acute respiratory diseases ( ard ) , asthma , conjunctivitis and dermatitis were obtained for the period of 2006 to 2010 , for children ages < 20 years old , for two large provinces around siz .
three exposure zones were defined on the basis of the distance from siz determined as : 5 , > 5 to 10 , 20 km to represent high , intermediate and control exposure zones , respectively .
age - specific and gender - specific monthly counts of visits were modelled using generalised additive models controlling for time trends .
the high and intermediate exposure zones were later combined together due to the similarity of associations .
exposure effect modification by age , gender and socioeconomic status ( ses ) was also tested.resultsliving within 10 km from siz showed a greater association with ard ( risk ratio ( rr)=2.5 ; 95% ci=2.3 to 2.7 ) , asthma ( rr=3.7 ; 95% ci=3.1 to 4.5 ) , conjunctivitis ( rr=3.1 ; 95% ci=2.9 to 3.5 ) and dermatitis ( rr=2.7 ; 95% ci=2.5 to 3.0 ) when compared with the control zone .
no differences in associations were found for gender and ses groups ; greater effects were noticed in the 14-year - old group for asthma.conclusionsthis is the first study conducted in oman to examine the health effects of a young population living near an industrial park .
we hope that these findings will contribute in future developments of environmental health policies in oman . |
idiopathic megacolon is a rare and poorly understood clinical condition that usually presents as refractory abdominal distension and constipation .
it includes idiopathic megarectum , idiopathic megacolon , or a combination of these disorders . among them , idiopathic megacolon without megarectum is the most uncommon disorder . in this case , we experienced the idiopathic megacolon with bowel dilatation only proximal to the splenic flexure .
one month before being referred to our hospital , abdominal distension and constipation developed and the severity of both increased with time .
the patient could not defecate and the abdominal pain increased despite fasting and frequent enemas .
because of the intractable symptoms , she was referred to our emergency unit for further investigation . the physical examination during the obstructive episodes revealed marked abdominal distension with hypoactive bowel sounds .
the patient denied a history of metabolic , neurological , cardiovascular or pulmonary disease , previous abdominal or pelvic surgery , or a history of abdominal cancer , inflammatory disease or trauma .
the laboratory testing showed a normal hemoglobin , hematocrit , white blood cell count , blood glucose , creatinine , and coagulation profile .
the serum electrolytes were also normal except hypokalemia was present with a level of 2.7 mmol / l ( normal range 3.5 - 5.1
the abdominal computed tomography ( ct ) scan showed bowel distension , and the transition zone was detected at the splenic flexure ( fig .
anorectal manometry showed a normal resting anal sphincter pressure , normal squeezing pressure during maximal voluntary effort , rectal sensory threshold , maximal tolerance volume ( 120 ml ) , and rectoanal inhibitory reflex was present .
the colon transit time was delayed ( total 135.6 hours , right colon 66 hours , left colon 56.4 hours , and rectosigmoid colon 13.2 hours ) . based on these findings
however , the abdominal distension and constipation were not improved . on the 19th day of hospitalization
, a small amount of loose stool was passed and the abdominal pain was relieved .
on the 22nd day of hospitalization , the frequency of loose stools increased up to 3 - 4 times / day .
oral feeding was started on the 27th day of hospitalization after checking the ct endoscopy ( fig .
the finding was marked dilatation of ascending and transverse colon at the level of distal transverse colon without obstructive lesion .
the patient was discharged on the 44th day of hospitalization on oral mosapride , itopride , domperidone , and lactitol ( fig .
after discharge , she did not have additional symptoms even after the medication was discontinued .
she was admitted to a local hospital for 20 days and sigmoidoscopic irrigation and enemas were performed there
severe abdominal distension was noted on physical examination and on the simple abdominal radiograph ( fig . 3a ) .
the laboratory testing was normal except for alt ( 119 u / l ) and ast ( 141 u / l ) .
bisacodyl / docusate was started on hospital day 2 . by hospital day 8 , loose stools were passed after a glycerin enema and the abdominal pain and distension were relieved . on hospital day 10 , a large amount of loose stool was passed and magnesium oxide , mosapride , domperidone , and colonlyte were started .
stool passage was increased to 7 - 15 times / day . on hospital day 16 , oral feeding was started and the alt and ast returned to normal . on hospital day 25 , the patient was discharged ( fig .
three months later , she visited the outpatient clinic and did not have abdominal distension or constipation without medication ( fig .
megacolon can be observed in hirschsprung 's disease , with an infection ( chaga 's disease ) and in some disorders of the endocrine or central nervous system ( including spinal trauma and senility ) .
however , in the absence of an organic cause , it is referred to as idiopathic.1,2 idiopathic megacolon is a heterogeneous disorder including idiopathic megarectum , idiopathic megacolon , or a combination of these disorders.3 idiopathic proximal hemimegacolon is defined as megacolon only proximal to the splenic flexure.4 idiopathic proximal hemimegacolon is a very rare and therefore a poorly understood clinical entity .
the plausible explanations include abnormalities involving the extrinsic nerves , the enteric nerve plexus , or the intestinal smooth muscle , which could lead to gut dilatation and impaired motility.5 anal manometry is clinically useful in a few patients with chronic constipation , although it has potential value for the exclusion of hirschsprung 's disease , due to the demonstration of internal sphincter relaxation in response to rectal distension.6 the clinical features of patients with idiopathic proximal hemimegacolon are similar to those reported in idiopathic megacolon without megarectum in previous studies .
patients with idiopathic megacolon are older than patients with idiopathic megarectum . in this case , the patient experienced her first episode at 44-years of age . most patients present with constipation ; however , documented bowel habits are very variable , from 5 times per day to only once every 3 months .
our patient had abdominal distension and pain , normal intelligence , and no familial history .
delay in the colon transit is most prominent in the right colon area . in the rectosigmoid area , it is normal.2 - 4,7,8 our patient also showed delay in right colon transit .
the abnormal ast / alt levels were considered secondary to hepatocongestion induced by the megacolon .
the majority of patients can be successfully managed without surgery.2 for shortening bowel transition time and symptom relief , the prokinetic drugs like mosapride and itopride could be coadministrated.9 however , medical treatment may fail to alleviate symptoms in some patients,8 may be poorly tolerated , and must be continued lifelong to prevent the recurrence of symptoms .
megacolon proximal to the splenic flexure is referred to as idiopathic proximal hemimegacolon . here , we present the case of a 44-year - old woman with idiopathic proximal hemimegacolon that had 2 episodes over 4 years . | idiopathic proximal hemimegacolon is a disorder characterized by bowel dilatation proximal to the splenic flexure .
it is a very rare and therefore a poorly understood clinical entity .
this report describes a case of idiopathic proximal hemimegacolon in a 44-year - old woman .
the patient suffered from 2 episodes of constipation and bowel dilatation over 4 years and was successfully treated by medical therapy . |
road accidents and the related mass casualties are one of the challenges of the human society that threaten the health of people and impose huge costs on the states .
developed countries have the largest number of victims due to road accident and is considered as one of the main causes of death .
the degree of injuries and the frequency of major road accidents are to such extent that it is called the
according to the statistics , on average , 10% of the injuries lead to death and about 3,000 people are killed per day due to traffic accidents around the world .
similar to other human factors , the behavior of a driver is influenced by conscious and unconscious factors called cognitive - behavioral characteristics .
based on a study , driving errors ( i.e. human factors ) are responsible for 93% of traffic accidents , 34% due to operational deficiencies , and 12% due to vehicle malfunctions .
the results confirm reports that indicate the role of human factors ( individual or social ) in road accidents .
moreover , other studies have highlighted the role of driver s personality and visibility in driving behavior . according to past research studies , drivers that cause accident
are distinguished from other drivers by factors such as empathy , characteristics , stress level , and few other parameters .
an analysis of the road accidents in iran also revealed several factors for such occurrences .
showed that the contribution of human factors to road accidents is 97.5% and the role of human factors in causing an accident is 49% . in their research ,
hassanpour et al . found that human factors are one of the most important causes of driving accidents in iran ( more than 30% of accidents and 23% of driving losses ) .
moreover , refahi et al . stated that the role of attitude in driving behavior is more important than other aspects .
consistent with their opinion , there was an indirect association between variables such as personality traits and driving behavior .
in general , human factors that are involved in road accidents are categorized into two general classifications , namely cognitive - behavioral factors and behavioral factors . for the latter ,
behavioral corresponds to the contributing factors ( e.g. infringement of driving laws , driving at high speed while feeling dizzy and fatigued , drug or alcohol use ) in causing a road accident . while recognizing the adverse effect of road conditions and vehicle health in causing accidents , researchers have hypothesized that the effect of human factors is omitted and should be investigated .
the discarded factors are personality traits ( based on the big five personality traits ) , driving behavior , and mental disorders ( based on the diagnostic interview ) .
this study was conducted to evaluate the specific hypothesis that the cognitive - behavioral factors and mental disorders can predict road accidents .
the aim of this cohort study was to identify the psychological factors that influence road accidents .
in addition , we compared driving behavior and its subscales in two groups of drivers with accidents and those without road crash .
due to increased fatalities and an upsurge in road trips in iran , our country is also not immune from road accidents .
in this cohort study , 800 bus and truck drivers were recruited among those referred to imam sajjad hospital ( tehran , iran ) during october 2013 to may 2015 .
these drivers referred to the hospital for a drug test , as a medical pre - requisite for renewing their driver s license . as a profession , they were heavy vehicle drivers and were holding category c or d driver s license .
we intentionally targeted heavy vehicle drivers since they are in high - risk accident category compared to other drivers .
convenience sampling , a non - probability sampling technique , was used in this study .
initially , under the supervision of interviewers , the participants were encouraged to fill in the questionnaire forms .
this was followed by a semi - structured interview ( schizophrenia and affective disorders scale [ sads ] ) by a clinical psychologist to diagnose any mental disorders .
the questionnaires comprised of demographic questions , manchester driving behavior questionnaire ( mdbq ) , neo personality inventory , and smoking and substance abuse inventory .
the inclusion criteria were male drivers , holders of category c or d driver s license , age range 23 - 85 years and being referred to imam sajjad hospital .
the exclusion criteria were female drivers , suffering from severe mental disorders ( e.g. chronic psychosis or dementia ) , and illiteracy or uneducated drivers unable to understand the questions .
then , based on police accident reports , we identified those drivers that were involved in a driving accident due to human factors .
the demographic questions consist of participants age , education , history of driving , the amount of kilometers traveled per month , marital status , time taken to obtain driver s license , and the number of occurred accidents caused by human factors .
this scale was adjusted and compiled by rissen et al . in the psychology department of manchester university , manchester ,
it is based on the idea that errors and violations have different psychological reasons and correction methods ; hence , they should be discriminated by researchers .
the questions cover two different aspects ; the kind of behavior and the amount of risk posed to other drivers .
these behaviors are classified as follows :
behaviors that pose no risk to others and merely give a feel of comfort ( low - risk probability)behaviors that are likely to put others at risk ( moderate - risk probability)behaviors that certainly put others at risk ( high - risk probability ) .
behaviors that pose no risk to others and merely give a feel of comfort ( low - risk probability ) behaviors that are likely to put others at risk ( moderate - risk probability ) behaviors that certainly put others at risk ( high - risk probability ) .
obtained a correlation coefficient of 0.81 for errors and 0.75 for violations in another reliability research conducted on 80 drivers with a 7-week interval .
iliescu and srbescu reported that the reliability of mdbq factors is from 0.62 to 0.78 , which is satisfactory .
moreover , oreizi reported that the iranian version of mdbq has acceptable reliability and validity . in their study , the reliability of the factors ranged from 0.65 to 0.81 .
paul costa ( 1985 ) designed this questionnaire as a short form of the neo personality inventory .
each question inquired about one of the main five personality dimensions , including neuroticism ( n ) , extraversion ( e ) , openness to experience ( o ) , agreeableness ( a ) , and conscientiousness ( c ) , respectively .
each of the factors covered 12 questions and each scale scored from 0 to 48 .
the validity and reliability of the persian version of neo personality inventory have been confirmed in various studies .
yadollahi et al . noted that the short form of neo has acceptable reliability ( cronbach between 0.69 - 0.83 ) .
this questionnaire consists of 12 items , with five - point likert spectrum , and it surveys the dosage of the drugs , stimulants , hallucinogens , and alcohol during the past year .
clinical psychologists conducted the semi - structured interview to diagnose any mental disorder in drivers .
this interview evaluated 16 main psychology and psychiatric disorders and other psychiatric disorders , including psychiatric problems , depression , mania , hypomania , substance abuse and addiction , psychosis , anxiety disorders , antisocial personality , somatization disorders , suicidal thoughts , post - traumatic stress disorders ( ptsd ) , dissociative disorders , epilepsy disorder , alzheimer , and mental retardation .
simpson et al . reported that cohen s kappa coefficient of mania , hypomania and depression was calculated as 0.83 , 0.72 and 1 , respectively .
the diagnostic interviews were performed by clinical psychologists that had been educated in the field of diagnosis and treatment of mental disorders .
data were analyzed using the statistical package for social sciences ( spss ) version 18.0 .
in addition , descriptive statistics were used to demonstrate demographic data . to compare driving behavior and its subscales
the effective factors that contribute to road crashes were determined using multiple logistic regression analysis .
p values less than 0.05 were considered as statistically significant . to analyze the data ,
all variables including personality traits , mental disorders , and demographic characteristics that were collected through questionnaires and were likely to have an impact on road accidents were identified .
the logistic regression analysis was conducted for each variable in the univariable analysis and crude or was also estimated . in the next step , variables with p values
then , those variables that had a significant role in the model with p values<0.05 were interpreted with adjusted or .
in this cohort study , 800 bus and truck drivers were recruited among those referred to imam sajjad hospital ( tehran , iran ) during october 2013 to may 2015 .
these drivers referred to the hospital for a drug test , as a medical pre - requisite for renewing their driver s license . as a profession , they were heavy vehicle drivers and were holding category c or d driver s license .
we intentionally targeted heavy vehicle drivers since they are in high - risk accident category compared to other drivers .
convenience sampling , a non - probability sampling technique , was used in this study .
initially , under the supervision of interviewers , the participants were encouraged to fill in the questionnaire forms .
this was followed by a semi - structured interview ( schizophrenia and affective disorders scale [ sads ] ) by a clinical psychologist to diagnose any mental disorders .
the questionnaires comprised of demographic questions , manchester driving behavior questionnaire ( mdbq ) , neo personality inventory , and smoking and substance abuse inventory .
the inclusion criteria were male drivers , holders of category c or d driver s license , age range 23 - 85 years and being referred to imam sajjad hospital .
the exclusion criteria were female drivers , suffering from severe mental disorders ( e.g. chronic psychosis or dementia ) , and illiteracy or uneducated drivers unable to understand the questions .
then , based on police accident reports , we identified those drivers that were involved in a driving accident due to human factors .
the demographic questions consist of participants age , education , history of driving , the amount of kilometers traveled per month , marital status , time taken to obtain driver s license , and the number of occurred accidents caused by human factors .
this scale was adjusted and compiled by rissen et al . in the psychology department of manchester university , manchester ,
it is based on the idea that errors and violations have different psychological reasons and correction methods ; hence , they should be discriminated by researchers .
the questions cover two different aspects ; the kind of behavior and the amount of risk posed to other drivers .
these behaviors are classified as follows :
behaviors that pose no risk to others and merely give a feel of comfort ( low - risk probability)behaviors that are likely to put others at risk ( moderate - risk probability)behaviors that certainly put others at risk ( high - risk probability ) .
behaviors that pose no risk to others and merely give a feel of comfort ( low - risk probability ) behaviors that are likely to put others at risk ( moderate - risk probability ) behaviors that certainly put others at risk ( high - risk probability ) .
obtained a correlation coefficient of 0.81 for errors and 0.75 for violations in another reliability research conducted on 80 drivers with a 7-week interval .
iliescu and srbescu reported that the reliability of mdbq factors is from 0.62 to 0.78 , which is satisfactory .
moreover , oreizi reported that the iranian version of mdbq has acceptable reliability and validity . in their study , the reliability of the factors ranged from 0.65 to 0.81 .
paul costa ( 1985 ) designed this questionnaire as a short form of the neo personality inventory .
each question inquired about one of the main five personality dimensions , including neuroticism ( n ) , extraversion ( e ) , openness to experience ( o ) , agreeableness ( a ) , and conscientiousness ( c ) , respectively .
each of the factors covered 12 questions and each scale scored from 0 to 48 .
the validity and reliability of the persian version of neo personality inventory have been confirmed in various studies .
noted that the short form of neo has acceptable reliability ( cronbach between 0.69 - 0.83 ) .
this questionnaire consists of 12 items , with five - point likert spectrum , and it surveys the dosage of the drugs , stimulants , hallucinogens , and alcohol during the past year .
clinical psychologists conducted the semi - structured interview to diagnose any mental disorder in drivers .
this interview evaluated 16 main psychology and psychiatric disorders and other psychiatric disorders , including psychiatric problems , depression , mania , hypomania , substance abuse and addiction , psychosis , anxiety disorders , antisocial personality , somatization disorders , suicidal thoughts , post - traumatic stress disorders ( ptsd ) , dissociative disorders , epilepsy disorder , alzheimer , and mental retardation .
simpson et al . reported that cohen s kappa coefficient of mania , hypomania and depression was calculated as 0.83 , 0.72 and 1 , respectively .
the diagnostic interviews were performed by clinical psychologists that had been educated in the field of diagnosis and treatment of mental disorders .
data were analyzed using the statistical package for social sciences ( spss ) version 18.0 .
in addition , descriptive statistics were used to demonstrate demographic data . to compare driving behavior and its subscales
the effective factors that contribute to road crashes were determined using multiple logistic regression analysis .
p values less than 0.05 were considered as statistically significant . to analyze the data ,
all variables including personality traits , mental disorders , and demographic characteristics that were collected through questionnaires and were likely to have an impact on road accidents were identified .
the logistic regression analysis was conducted for each variable in the univariable analysis and crude or was also estimated . in the next step ,
variables with p values < 0.2 were selected and entered into the multiple logistic regression analysis .
then , those variables that had a significant role in the model with p values<0.05 were interpreted with adjusted or .
their age ranged from 24 to 81 years with an average age of 46.711.17 years ( meansd ) .
the majority of the 654 drivers ( 81.7% ) were married , 730 ( 91.2% ) had a secondary school education or less , and 70 ( 8.8% ) were college graduates .
summary of demographic information from 800 bus and truck examinees driving history : number of years of driving per person table 2 shows the mean , standard deviation , and comparison of the driving behavior and its subscales between the two groups of drivers with road accidents and without road crash .
the t test analysis indicated that the two groups differed significantly in terms of driving behavior and all its dimensions such as slips , deliberate violation , laps error , and unintentional violation ( p<0.05 ) .
the mean , standard deviation , and comparison of driving behavior and its subscales between the two groups of drivers with accidents and without road crash driving behavior : scores of driver s driving behavior the findings of this study revealed that age reduced the risk of accident occurrence ( positive effect ) , indicating that by per year increase in age , the odds of accident occurrence was reduced by 0.01-fold .
in addition , we found that driving history also reduced the risk of accidents ( positive effect ) , meaning that by per year increase in driving history , the odds of accident occurrence were reduced by 0.05-fold .
based on the multiple logistic regression interpretations ( multivariable analysis ) , accident history had no impact on the risk of accident occurrence ( table 3 ) .
multiple logistic regression - independent predictors of road accident in truck and bus drivers ( n = 800 ) the results revealed that depression , in three levels of minor , mild and major , increased the odds of road accidents by 2.4-fold . furthermore , with regard to anxiety disorders , particularly obsession disorder , the results revealed that this disorder increased the odds of road accidents by 2.7-fold .
ptsd and driving behavior were not significant parameters in the equation ; and among the personality traits , neuroticism was more likely to increase crash probability by 1.1-fold , but other personality factors did not seem to have a significant effect on the risk of accident occurrence . moreover , since none of the drivers mentioned any disorders , such as smell or vision delusion , psychosis symptoms , epilepsy , mania , hypomania , panic disorder , phobia , fugue , or other mental problems , we therefore did not enter any of these disorders into the equation .
the aim of this cohort study was to identify the possible impact of personality traits , some mental illnesses , and driving behavior of bus and truck drivers on the risk of road accidents .
after two years of follow - up , we surveyed those samples that had road accidents due to human factors .
based on the results of this study , older age reduces the occurrence of accidents ( positive effect ) , which means that per year of age the odds of accident occurrence is reduced by 0.01-fold .
for example , in a substantial research done by heidari et al . , they reported that there was a significant relationship between age and all the errors and infringements of driving .
the risk of driving violations is higher in youths aged 18 - 25 years old , as this age range is one of important risk factors for road accidents . on the other hand , mehmandar et al .
argued that older age and other demographic properties such as a university degree and female gender seem to be the most vital risk factors in road accidents that lead to fatalities in iran .
clearly , driving is a complex activity and includes cognitive , visual , and bodily processes .
hence , driving could be adversely affected by factors such as age and mental illness that can affect cognitive processes involved with driving .
therefore , it can be stated that older people are more prudent and less likely to be involved in a road accident .
moreover , the ability to drive safely and skillfully is a process that takes 8 to 9 years .
the results of our study suggest that it will take several years for a novice driver to turn into an experienced driver . to clarify the relationship between age and reduction in the occurrence of traffic accidents
, we can state that older people are more experienced and cautious than young drivers .
awareness of mental and physical limitations is more realistic in older drivers , so they exhibit self - control behavior that leads to a better risk estimation while driving .
the results revealed that driving history could reduce the likelihood of accidents ( positive effect ) , meaning that as driving experience is enhanced every year and as a driver reaches an older age , the chance of an accident will reduce . in other research studies , it is stated that compared with a history of car accidents , the duration of a driver s license is more associated with the reduced risk of accidents due to human factors ; and more experience is associated with reduced accidents .
based on the results of a study , having visited a psychiatrist or psychologist did not affect the risk of accident occurrence .
this result is in contrast with those of previous research studies ; for instance , a study noted that tired , mindless , careless and anxious drivers experience difficulty while driving and they sometimes exhibit risky driving behavior . in another study , it was mentioned that patients with psychiatric disorders had twice as many accidents compared with a control group .
the inconsistency between our results and those of the previous studies could be due to the utilization of different sample groups and diagnostic tools . in iran , referring to a psychologist or psychiatrist is occasionally considered taboo , which in turn could negatively affect the results .
the preliminary results revealed that depression in three levels of minor , major and mild was a predictor of an accident and increased the odds of accidents by as much as 2.4-fold . in a similar research
, it was found that risky driving behavior is associated with sensation seeking , lack of tolerance , stress intolerance , impulsivity , violence , emotional instability , adventure , depression and pessimistic thoughts .
another research found a positive and robust correlation between depression and risky driving behavior , especially in injury accident and driving while intoxicated .
da silvia - junior et al . noted that truck drivers were more susceptible to depression in comparison with the general population and depression prevalence among them was 13.6% .
other studies mentioned that patients who suffer from psychiatric illnesses ( such as depression or anxiety ) have a double accident versus a control group .
the results revealed that mania and hypomania , as well as a history of psychiatric problems , did not increase or decrease the risk of accidents .
therefore , it can be concluded that these results arise from the lack of proper sample size or the absence of mania or hypomania symptoms in the sample group . in anxiety disorders spectrum ,
especially obsession disorder , the results demonstrated that obsession disorders increased the odds of accidents by 2.7-fold in truck and bus drivers .
previous reports demonstrated that a significant correlation was found between neurosis such as anxiety and depression with road crashes .
however , in other research , significant differences were reported between the scores of paranoid thoughts , obsessive and compulsive behavior , and interpersonal sensitivity in both groups of drivers with and without road accidents .
asghari et al . reported that there was a negative correlation between anxiety and driving psychology .
overall , it can be assumed that mental health is one of the variables that has been investigated in aggressive behaviors of drivers .
it is believed that persons , who suffer from mental disorders , may jeopardize the health of themselves and others with their driving behavior .
based on our findings , among the personality traits , only neuroticism increased the risk of accidents and other personality traits were not significant in the logistic regression equation .
these findings are consistent with studies by wang , rau , and solvency . they emphasized the role of vision and personality factors in road accidents .
another research also showed a positive correlation between the neuroticism index with a variety of errors and illegal acts in driving behavior , which confirms the results of this study .
moreover , a large study , which was conducted in 34 countries indicated that there was a negative correlation between neuroticism and traffic fatalities . in another study
, no positive relationship was found between aggressive driving and extroversion or sensation seeking . on the other hand , fyhri and
is also negatively associated with risky driving or is correlated with aggressive driving , which is inconsistent with our results .
the inconsistency between our results and those in the above - mentioned studies could be due to different populations , personality types , and the effect of other variables such as age , environmental factors , and history of driving . in one of the studies ,
the participants were young drivers whereas in our study the drivers were selected from all age ranges , which could have affected our results .
it should be noted that the complexity of human behavior stems from various factors such as cultural , educational , economic , social factors and mental health , which can affect the driving behavior of truck and bus drivers and may play an essential role in shaping their behavior .
first , data were collected over a very short period of time ( 2 years ) and administering the questionnaires ( mdbq and neo ) had certain constraints .
second , the procedure for selecting the samples did not allow us to generalize the results to other populations such as those holding category a and b driver s licenses .
the two main factors in the incidence of traffic accidents are the human factors and patterns of driving behavior . in this study , it was observed that the underlying factors of traffic accidents were human , cognitive , and behavioral factors such as mental illness , personality , or the age of drivers . using the results of this study
, we can control the percentage of road accidents in drivers holding category d and c driver s licenses .
in addition , by introducing necessary changes in the process of obtaining a driver s license ( e.g. performing a psychological test ) we can guide drivers to shape a safe driving behavior . the strengths of this study were its cohort study design and diagnostic interview of all participants to diagnose mental disorders .
since it has yet to be established whether the quality of road and vehicles are the cause of road crashes , researchers need to conduct longitudinal research on road accidents and perform the psychological tests precisely . | background : the aim of this study was to evaluate the effect of variables such as personality traits , driving behavior and mental illness on road traffic accidents among the drivers with accidents and those without road crash.methods:in this cohort study , 800 bus and truck drivers were recruited .
participants were selected among drivers who referred to imam sajjad hospital ( tehran , iran ) during 2013 - 2015 . the manchester driving behavior questionnaire ( mdbq ) , big five personality test ( neo personality inventory ) and semi - structured interview ( schizophrenia and affective disorders scale ) were used .
after two years , we surveyed all accidents due to human factors that involved the recruited drivers .
the data were analyzed using the spss software by performing the descriptive statistics , t - test , and multiple logistic regression analysis methods .
p values less than 0.05 were considered statistically significant.results:in terms of controlling the effective and demographic variables , the findings revealed significant differences between the two groups of drivers that were and were not involved in road accidents . in addition , it was found that depression and anxiety could increase the odds ratio ( or ) of road accidents by 2.4- and 2.7-folds , respectively ( p=0.04 , p=0.004 ) .
it is noteworthy to mention that neuroticism alone can increase the odds of road accidents by 1.1-fold ( p=0.009 ) , but other personality factors did not have a significant effect on the equation.conclusion:the results revealed that some mental disorders affect the incidence of road collisions .
considering the importance and sensitivity of driving behavior , it is necessary to evaluate multiple psychological factors influencing drivers before and after receiving or renewing their driver s license . |
insertion sequences ( is ) are short prokaryote transposable elements ( tes ) characterized by a simple structure , often consisting of a single transposase - encoding open reading frame flanked by terminal inverted repeats .
because their proliferation can induce variation in genome size , activate and inactivate gene expression and trigger various types of genomic rearrangements , is have a profound influence on prokaryote genome evolution .
the general lack of congruence between is and host phylogenies , together with their overall patchy distribution among hosts , suggest that is distribution among prokaryotes is strongly shaped by horizontal transfer .
unlike is , bacteriophages and plasmids encode the enzymatic machinery allowing them to move between cells .
these two types of mobile genetic elements ( mges ) have traditionally been considered as the main vectors of is horizontal transfer .
however the relative contribution of phages and plasmids to is horizontal transfer has never been thoroughly evaluated .
we recently reported a comprehensive survey of the abundance and distribution of is elements integrated in 900 phage and plasmid annotated genomes available in the aclame database .
this study revealed that while over half of plasmids contain at least one is ( with a density of one copy every 19 kb on average ) , the vast majority ( 92% ) of phages are completely devoid of is insertion ( one copy every 346 kb on average ) .
we further showed that the density of deleterious insertion sites is significantly higher in phages than in plasmids .
consequently , acquisition of is elements is more strongly counter - selected in phages than in plasmids .
based on these results , we concluded that phages are less tolerant to is insertions than plasmids , i.e. , phages have a lower cargo capacity than plasmids .
therefore , while our analysis confirmed the classically held view that plasmids are efficient vectors of is horizontal transfer , we concluded that phages may be rather poor vectors of is horizontal transfer among prokaryotes .
if phages indeed do possess a lower cargo capacity than plasmids do , then we expect the distribution pattern observed for is elements to also hold for other tes .
transposons are certainly the most thoroughly studied bacterial tes . they comprise elements with various modes of mobility :
( 1 ) composite ( formerly class i ) transposons are genetic sequences flanked by is elements which use the is transposase for their mobility , ( 2 ) unit transposons ( formerly type ii / tn3 family ) which use specific transposase / resolvase genes for their mobility , and ( 3 ) conjugative transposons / ices which can transfer from a bacterial cell to another through conjugation .
transposons are often found in plasmids . on the contrary , to our knowledge , there has been no report of a transposon being carried by an active bacteriophage .
the distribution of transposons in plasmids and phages therefore seems fully consistent with higher selective constrains on phages compared with plasmids .
mobile introns are other bacterial tes which are able to self - splice from the transcript they are inserted in .
they include two unrelated types of elements : group i introns ( which move through a homologous recombination process termed homing ) and group ii introns ( which move through reverse - transcription and integration of an rna intermediate in a process termed retrohoming ) .
group ii introns are widespread in the bacterial world and frequently observed within plasmid sequences . to our knowledge ,
the distribution of group ii introns in plasmids and phages thus apparently reflects that of is elements .
recent studies suggest that group ii intron insertions may be deleterious for hosts despite their splicing ability .
while the greater tolerance of plasmids to group ii introns is likely due to lower selective constraints acting on their genomes compared with phages genomes , the presence of some group ii introns in plasmids may also be explained by their insertional preferences .
for example , the well - studied group ii intron ll.ltrb performs specific retrohoming into relaxase genes , which are parts of the plasmid mobility machinery .
analogously , members of the giic - attc intron class target integrons themselves often integrated within plasmids . finally , the targeting of is elements by various group ii introns may also account for group ii intron preferential presence in plasmids compared with phages . in contrast with group ii introns , group i introns are preferentially found in phages rather than plasmids .
many group i introns have indeed been detected in phage sequences but to our knowledge , only two copies have been reported in a plasmid .
furthermore , these two plasmid - borne group i introns are inserted within a prophage which is itself inserted into the plasmid genome .
therefore , there is as yet no evidence that a group i intron has jumped into a plasmid genome .
the pattern of group i intron distribution in mges is puzzling as it is contradictory to that observed for other tes , but it may be explained by the specific mobility mechanism used by group i introns . indeed , group i introns move from an intron - containing locus to a highly similar intron - free locus ( generally another allele of the same locus ) through a recombination - mediated process .
the break is then repaired by the host homologous recombination machinery , which uses the intron - carrying allele as a template .
as homologous recombination proceeds only on highly similar regions , the spread of group i introns relies on their ability to invade conserved regions .
accordingly , chromosomal group i introns are usually found in trna and rrna genes or other essential genes , while most phage - borne group i introns are found in genes important for phage biology . in this context
, the absence of group i intron in plasmids might simply reflect the fact that these site - specific elements never evolved to target plasmid genes . in sum , this brief overview of the distribution of the main bacterial tes in plasmids and phages globally supports our previous observations on is elements .
it is fully consistent with higher selective constraints applying to phage genomes compared with plasmid genomes .
in addition to the density of deleterious insertion sites , other factors may also contribute to the basis for strong negative selection against te insertions in phages and plasmids , and thus their cargo capacity .
for example , growth rates of plasmid - carrying bacteria are negatively correlated with plasmid genome size .
however , the decrease in growth rate induced by plasmid genome size increase is negligible compared with that induced by highly expressed genes .
thus , a small plasmid with a single highly expressed gene may be much more counter - selected than a large plasmid with many lowly expressed tes .
by contrast , genome size is a much more salient constraint in phages because capsids ( i.e. , phage container particles ) can not package more than 105106% of the wild type genome size . with a median size of 42 kb for phage genomes
, this means that half of phage genomes can not package more than 2.5 kb of additional sequence , which translates into a couple of is elements or one group ii intron , at most .
thus , constraints on genome size also argue for a larger cargo capacity for plasmids relative to phages .
however , whatever its strength , purifying selection has to be efficient in order to effectively purge mge genomes from deleterious te insertions .
population genetics theory indicates that selection efficiency depends on effective population size : the higher the effective population size , the more efficient the selection .
phages are the most numerous organisms on earth and their effective population size is presumably enormous . thus ,
in addition to the fact that their genome contains a higher proportion of deleterious sites , the efficiency of purifying selection in phages is likely to be much higher than in plasmids : for an equally deleterious insertion site , purifying selection is expected to remove a te quicker from a phage population than from a plasmid population . on the other hand , many more low - frequency neutral te insertions are expected in ( large ) phage populations than in ( smaller ) plasmid populations . even though the lifespan of individual insertions is very short
, they may collectively represent a dynamic reservoir of tes that might be transferable between bacterial cells .
the magnitude of such a phenomenon is currently difficult to estimate , but the availability of an increasing amount of metagenomic data is likely to provide new insights into this question in the near future .
if this hypothesis was verified , it would argue for a potentially more important role of phages in the horizontal transfer of tes than the current evidence suggests .
nevertheless , another population genetics parameter to keep in mind for a horizontal transfer to be successful is not only vector fitness , but also bacterial host fitness .
indeed , a large number of potential transfers initiated by phages simply can not lead to successful te horizontal transfers because they result in the death of the bacterial hosts . on the contrary
another factor that might affect the efficiency with which phages and plasmids can shuttle tes between bacteria is the breadth of mge host range .
while a substantial number of broad host - range plasmids and phages have been characterized , the majority of plasmids and phages are believed to have a relatively narrow host range and to be capable of transferring genetic material only between closely related species of the same genus .
this trend is indeed consistent with the observation that horizontal transfer of both is and non - is dna is more frequent between closely related bacteria than between distantly related bacterial lineages .
overall , the available data on mge host range do not seem to indicate any dramatic difference between typical phage and plasmid host ranges .
however , studies of mge host ranges remain relatively scarce and are often plagued with various pitfalls and limitations .
a method was recently developed to predict plasmid host range based on genomic signatures expected to be shared ( or not ) between plasmids and bacterial chromosomes , depending on plasmid host range breadth .
this method , which exclusively relies on sequence data , was first validated on various plasmids belonging to six well - studied incompatibility groups ( inc.f , inc.h , inc.i , inc.n , inc.p and inc.w ) and was then applied to other less studied plasmids ( inc.q , inc.u , proma , inc.a / c and inc.p-9 ) for which host range was mostly inferred to be wide . given the rate at which new mges and bacterial genomes are currently added to public databases , it will certainly help to better delineate the host range of numerous mges .
it is however important to keep in mind that simple entry of a te - carrying mge into a new bacterial cell is potentially sufficient for te horizontal transfer to happen . in other words
, mges can facilitate te horizontal transfer even in the absence of successful phage replication or plasmid maintenance .
therefore , the host range relevant to a particular mge ( i.e. , the number of bacterial species in which a phage can successfully replicate or in which a plasmid can be maintained ) might be narrower than the host range truly relevant to te horizontal transfer .
our comparison of is abundance in phages and plasmids has provided the first formal test of the relative contributions of phages and plasmids to is horizontal transfer between bacterial cells , suggesting that plasmids are better vectors of is horizontal transfer than phages .
this result seems to also hold for other tes , such as transposons and group ii introns .
in fact , the better capacity of plasmids to shuttle tes compared with phages is probably not due to an intrinsic property of tes , as a recent study showed that this trend seems to apply to all types of dna sequences .
we have summarized a number of potential underlying causes for the differential implication of phages and plasmids in the horizontal transfer of tes .
while strength and efficiency of selection are clearly major players in this game , a number of other parameters ( e.g. , low - frequency insertions in phages and mge host range ) may have a non - trivial impact as well .
however , the actual magnitude of the different factors currently remains difficult to evaluate with accuracy .
there is no doubt that the ever - increasing amount of genomic data being made available will soon allow for refined estimates of these parameters . | horizontal transfer of transposable elements ( tes ) plays a key role in prokaryote genome evolution .
most tes do not encode the enzymatic machinery allowing them to transfer between host cells and it is widely assumed in the literature that horizontal transfer of prokaryote tes is mediated by other mobile genetic elements such as phages and plasmids . in a recent study , we have shown that phages are less tolerant to insertion sequences ( is , the most frequent class of prokaryote tes ) and therefore have a lower cargo capacity than plasmids . consequently , while our analysis confirmed the crucial role of plasmids as efficient vehicles of is horizontal transfer , we concluded that phages are unlikely to efficiently shuttle is elements between prokaryotes .
here , we discuss whether or not the distribution pattern observed for is elements in phages and plasmids also holds for other tes , such as transposons and mobile introns .
we also further explore various factors that may impact the relative capacity of phages and plasmids to mediate te horizontal transfer among prokaryotes . |
chronic obstructive pulmonary disease ( copd ) is an airway inflammatory disease caused by inhalation of toxic particles , mainly cigarette smoking , and is now accepted as a disease associated with systemic characteristics.1 nutritional status is one of them .
twenty - six percent to 70 percent of copd patients with the disease have malnutrition.2,3 in the global initiative for chronic obstructive lung disease , which was revised in 2006 , however , the malnutrition is considered to be developed only in moderate or severe patients.4 we often see smokers who do not fulfill the copd criteria on spirometry test ( forced expiratory volume in 1 second [ fev1]/forced vital capacity [ fvc ] < 70% ) but who suffer from copd - like symptoms ( eg , cough , sputum , dyspnea on effort ) and/or have copd - like findings on chest x - ray and/or computed tomography.5 there are few reports that have evaluated nutritional status of copd - like symptomatic elderly smokers compared with elderly copd patients or healthy elderly . in the current study we compared anthropometrics , dietary intake , and nutritional status among elderly smokers with and without copd diagnosis and healthy elderly controls .
thirteen clinically diagnosed copd patients , ten smokers with copd - like symptoms but with fev1/fvc > 70% , all of whom were current smokers with at least a 40 pack - year smoking history , and 27 never - smoking healthy controls were enrolled .
inclusion criteria for the copd group and the symptomatic smokers ( sysm ) group were having copd - related symptoms ( dyspnea on effort , cough , sputum ) , regardless of the existence of copd - related image findings ( chest x - ray hyperinflation , flattened diaphragm , low attenuation area , or bullous changes on high - resolution computed tomography for chest ) , no medication for copd , and no participation in a rehabilitation program for the last 6 months .
patients were excluded if they had exacerbation , respiratory infection in the last 4 weeks , a history of chronic liver or renal failure , electrolyte abnormality , malignancy , insulin - dependent diabetes mellitus , usage of systemic corticosteroids , atopy , self - reported asthma , or reversibility > 12% of airway obstruction after administration of a 2-agonist for asthma or clinically apparent heart failure .
atopic status was assessed by the negative history and the negative immunoglobulin e radioallergosorbent test to 26 common allergens . due to the aforementioned exclusion criteria ,
65 subjects were excluded ( 58 for respiratory infection in the last 4 weeks , 13 for asthma diagnosis or complications , three for malignancy , one for heart failure ) .
the study was approved by the scientific ethical committee of kawasaki medical school hospital , and all patients gave informed consent .
all subjects underwent a medical history taking and medical examination by an experienced pulmonologist , spirometry for measuring fev1 and fev1/fvc before and after bronchodilation , and biochemical blood tests , including analysis for albumin , prealbumin and transferrin , electrolytes , and renal and liver function .
anthropometry factors , resting energy expenditure ( ree ) , and diet intake were evaluated .
lung function was evaluated using a dry spirometer ( chestac-33 ; chest mi , tokyo , japan ) , which met the 1994 american thoracic society recommendations for diagnostic spirometry .
three trials were performed , and the two higher inspiratory capacity values had to agree within 5% or 60 ml.6 the patients with fev1/fvc < 70% were classified to the copd group , and patients with fev1/fvc > 70% to the sysm group .
for calculation of spirometric lung age we used the japanese respiratory society s normal prediction equation for fev1 .
the measured fev1 and the patient s height were substituted to calculate the functional age , which has been termed the lung age as follows : ( 0.036 height ( cm ) 1.178 fev1.0 ( l))/0.028 . when the result was more than 95 ,
lung age was set to 95 years old.79 height , weight , percentage ideal body weight , body mass index ( bmi ) ( calculated as weight / height squared ) , body fat percentage measured by bioelectrical impedance analysis ( ss-103 bio impemeter , sekisui , osaka , japan ) , midupper arm muscle circumference ( amc ) , tricep skin fold thickness ( tsf ) , and ree were measured .
predicted values were quoted from japanese standardized data.10 skin fold measurements were taken from the triceps and shoulder blade ( subscaplar ) .
the body fat percentage was estimated using the equations of brozek et al.11 the equations for predicting body surface area ( bsa ) , body density ( d ) , and body fat percentage were as follows : bsa = 72.46 height ^ 0.725 body weight ^ 0.425 ,
d = 1.0923 0.000514 ( the total of skin fold thickness of the back of the arm and shoulder blade bsa/10,000/body weight ( kg ) 100 , body fat percentage ( % ) = ( 4.570/d 4.142 ) 100 . for ree measurements , an indirect calorie meter was used ( deltatrac ; datex corp , helsinki , finland ) . all food , except for water or tea , was forbidden from 21:00 on the previous day .
the ree was measured awake , in a resting state , and while in a supine position between 07:00 and 08:00 .
diet intake in a different 24 hours from the examination day was evaluated using a food value calculating software based on japanese standard nutrition allowance surveillance data for intake energy , protein , fat , carbohydrate , vitamins , phosphorus , calcium , potassium , magnesium , iron , fiber , and sodium.11 data are presented as mean or median ( 95% confident interval [ ci ] ) .
the statistical significance of differences among the three groups ( control , sysm , and copd groups ) was estimated with the kruskal - wallis test and analysis of variance .
the analysis was performed using statistical software ( stat view 4.0 ; abacus concepts co , ltd , baltimore , md ) and p < 0.05 was considered significant .
thirteen clinically diagnosed copd patients , ten smokers with copd - like symptoms but with fev1/fvc > 70% , all of whom were current smokers with at least a 40 pack - year smoking history , and 27 never - smoking healthy controls were enrolled .
inclusion criteria for the copd group and the symptomatic smokers ( sysm ) group were having copd - related symptoms ( dyspnea on effort , cough , sputum ) , regardless of the existence of copd - related image findings ( chest x - ray hyperinflation , flattened diaphragm , low attenuation area , or bullous changes on high - resolution computed tomography for chest ) , no medication for copd , and no participation in a rehabilitation program for the last 6 months .
patients were excluded if they had exacerbation , respiratory infection in the last 4 weeks , a history of chronic liver or renal failure , electrolyte abnormality , malignancy , insulin - dependent diabetes mellitus , usage of systemic corticosteroids , atopy , self - reported asthma , or reversibility > 12% of airway obstruction after administration of a 2-agonist for asthma or clinically apparent heart failure .
atopic status was assessed by the negative history and the negative immunoglobulin e radioallergosorbent test to 26 common allergens . due to the aforementioned exclusion criteria ,
65 subjects were excluded ( 58 for respiratory infection in the last 4 weeks , 13 for asthma diagnosis or complications , three for malignancy , one for heart failure ) .
the study was approved by the scientific ethical committee of kawasaki medical school hospital , and all patients gave informed consent .
all subjects underwent a medical history taking and medical examination by an experienced pulmonologist , spirometry for measuring fev1 and fev1/fvc before and after bronchodilation , and biochemical blood tests , including analysis for albumin , prealbumin and transferrin , electrolytes , and renal and liver function .
anthropometry factors , resting energy expenditure ( ree ) , and diet intake were evaluated .
lung function was evaluated using a dry spirometer ( chestac-33 ; chest mi , tokyo , japan ) , which met the 1994 american thoracic society recommendations for diagnostic spirometry .
three trials were performed , and the two higher inspiratory capacity values had to agree within 5% or 60 ml.6 the patients with fev1/fvc < 70% were classified to the copd group , and patients with fev1/fvc > 70% to the sysm group . for calculation of spirometric lung age we used the japanese respiratory society s normal prediction equation for fev1 .
the measured fev1 and the patient s height were substituted to calculate the functional age , which has been termed the lung age as follows : ( 0.036 height ( cm ) 1.178 fev1.0 ( l))/0.028 . when the result was more than 95 ,
height , weight , percentage ideal body weight , body mass index ( bmi ) ( calculated as weight / height squared ) , body fat percentage measured by bioelectrical impedance analysis ( ss-103 bio impemeter , sekisui , osaka , japan ) , midupper arm muscle circumference ( amc ) , tricep skin fold thickness ( tsf ) , and ree were measured .
predicted values were quoted from japanese standardized data.10 skin fold measurements were taken from the triceps and shoulder blade ( subscaplar ) .
the body fat percentage was estimated using the equations of brozek et al.11 the equations for predicting body surface area ( bsa ) , body density ( d ) , and body fat percentage were as follows : bsa = 72.46 height ^ 0.725 body weight ^ 0.425 , d = 1.0923 0.000514 ( the total of skin fold thickness of the back of the arm and shoulder blade bsa/10,000/body weight ( kg ) 100 , body fat percentage ( % ) = ( 4.570/d 4.142 ) 100 . for ree measurements , an indirect calorie meter was used ( deltatrac ; datex corp , helsinki , finland ) . all food , except for water or tea , was forbidden from 21:00 on the previous day .
the ree was measured awake , in a resting state , and while in a supine position between 07:00 and 08:00 .
diet intake in a different 24 hours from the examination day was evaluated using a food value calculating software based on japanese standard nutrition allowance surveillance data for intake energy , protein , fat , carbohydrate , vitamins , phosphorus , calcium , potassium , magnesium , iron , fiber , and sodium.11
data are presented as mean or median ( 95% confident interval [ ci ] ) .
the statistical significance of differences among the three groups ( control , sysm , and copd groups ) was estimated with the kruskal - wallis test and analysis of variance .
the analysis was performed using statistical software ( stat view 4.0 ; abacus concepts co , ltd , baltimore , md ) and p < 0.05 was considered significant .
the mean spirometric lung age of the control group was no different from their mean real age ( 80.2 [ 95% ci : 75.3 to 85.1 ] vs 74.7 [ 70.3 to 79.1 ] ) .
symptomatic smokers had significantly lower fvc and % fvc than the control group ( p < 0.001 , respectively ) .
the sysm group had significantly lower weight , bmi , percentage ideal body weight , body fat percentage , amc , and tsf than the control group did ( p < 0.01 each ) , and all of them were similar to the copd group .
albumin , prealbumin , and transferrin in the sysm and copd groups were significantly lower than those in the control group ( p < 0.02 each ) .
energy , vitamins ( a , b1 , b2 , and c ) , calcium , iron , fiber , and sodium were significantly lower in the sysm group than in the control group ( p < 0.05 each ) , and they were similar to the copd group , respectively . in total , fev1% predicted was positively correlated with iron , calcium , vitamin a , and vitamin b1 intake ( p < 0.03 each ) . in the sysm and control groups , fev1% predicted was not correlated with any factors . in the copd group ,
fev1% predicted was positively correlated with vitamin a ( p = 0.03 ) and iron ( p = 0.03 ) intake .
we have found that elderly smokers who are symptomatic but who do not fulfill the copd diagnostic criteria have nutritional deficits that may be related to insufficient energy intake similar to that seen in copd patients . from the late 1950s , it has been observed that many copd patients have low body weight , which suggests it as an independent predictor for the mortality of the disease.1217 subjects with copd are known to have elevated respiratory consumption energy and ree compared with healthy persons , and they intake less and thereby develop malnutrition or cachexia.18,19 our study showed the nutritional similarities between the copd patients who are known to be very sick and the symptomatic patients whom the clinicians may not recognize as being really sick
. the lower levels of bmi , tsf ( an indicator of body fat deposition ) , amc ( an indicator of muscle volume ) , albumin , prealbumin , and transferrin in the sysm group than in the healthy control group were similar to those of the copd patients . however , ree levels were no different among the groups .
the intake of energy , vitamins , calcium , iron , fiber , and sodium was significantly decreased in the sysm and copd subjects compared with the healthy controls .
this may suggest that the nutrition decline of the subjects was not caused by ree increase but possibly by intake insufficiency . in the sysm group , protein and
a study reported that a high carbohydrate diet accelerates the production of carbon dioxide and respiratory quotient.20 therefore , a lower carbohydrate diet might prevent or improve subjects respiratory situation .
diet education may be important.21 in the study , iron and vitamin a intake was correlated with fev1 predicted only in copd groups , not in the sysm or control groups .
there are few reports about the iron nutrition effect on lung function.22 for vitamin a and lung function , respiratory cell damage , as provoked by benzo[a]pyrene , which is present in cigarette smoke,23 decreases the uptake of vitamin a into lung cells24 and leads to a local vitamin a deficiency of the lung tissues.25 these data suggest that in patients with mild copd , a local vitamin a deficiency can occur that induces pathophysiologic consequences such as narrowing of the lumina and loss of distensibility of the airways .
nutritional factors ( iron and vitamin a ) might be useful for early detection of copd and general worsening .
the expected positive and negative rates were set as 80% and 40% , and for comparison of the difference of factors were set as half or double .
although it is known that sarcopenia is the loss of muscle mass that happens to everyone with advancing age , our subjects were age matched , and the mean age was no different among the groups .
previous studies have already demonstrated that elderly copd patients have higher ree compared with controls , but the subjects with moderate or more severe copd , in contrast , in the smokers group in our study , did not have fev1/fvc < 70%.16
elderly smokers with copd - like symptoms have insufficient energy intake , even if they do not have diagnosed copd . | background and objectivewhereas nutrition deficits are recognized as an expression of systemic inflammation in the elderly with diagnosed chronic obstructive pulmonary disease ( copd ) , if they occur in symptomatic elderly smokers , unfulfilled copd criteria are not confirmed.methodsrespiratory function , anthropometry assessment , and diet intake evaluation of 13 copd patients ( copd group ) , ten symptomatic elderly smokers ( sysm group ) , and 27 healthy volunteers ( control group ) were compared .
all were 70 years old or older.resultsthe sysm group had lower body weight , body mass index , percentage ideal body weight , body fat percentage , arm muscle circumference , tricep skin fold thickness , serum albumin , prealbumin , and transferrin than the control group and were similar to the copd group ( p < 0.05 each and nonsignificant each ) . resting energy expenditure was no different among the groups .
intake of energy , vitamins ( a , b1 , b2 , and c ) , calcium , iron , fiber , and sodium was also lower in the sysm group than in the control group ( p < 0.05 all ) and was similar to the copd group.conclusionelderly smokers who are symptomatic but who do not fulfill the copd diagnostic criteria have nutritional deficits related to insufficient energy intake that are similar to those seen in copd patients . |
in 1932 , crohn , ginzburg and oppenheimer first presented the clinical and pathologic features of a new disease which affected the terminal ileum .
they described the disease as granulomatous inflammation associated with thickening of the bowel wall , and called the condition terminal ileitis .
it later became apparent , however , that any region of the gastrointestinal fract from the mouth to the anus could be affected , so its name was changed from regional ileitis to regional enteritis or crohn s disease .
interest in this disease has increased recently , because its incidence and prevalence have greatly increased in western countries since the beginning of the sixties .
the disease is characterized by its insidious onset with diarrhea , intermittent abdominal pain , general malaise , loss of weight and anemia ; by its indolent , prolonged and variable course ; by its diversity of clinical features ; by its perianal and sytemic complications ; and its remarkable tendency to recur after resection of the involved intestine . crohn s disease is quite rare in korea .
it is not well understood why the disorder is rare in korea , but diarrhea of uncertain etiology and intestinal tuberculosis are common among koreans , and there is a possibility that the true incidence of crohn s disease have been masked .
the subjects were 45 patients with crohn s disease , including 23 cases at the author s of the hospital , that had been reported in korea during the period of 34 years from 1852 to 1985 .
nearly all of the cases were diagnosed having crohn disease after surgical intervention and subsequent pathologic exam of the resected specimens because they had initially appeared to have apparent surgical problems such as colon cancer , intestinal perforation , intestinal obstruction or acute appendicitis . on a retrospective basis ,
45 patients were reviewed and the following details were recorded : sex , age of onset , site of macroscopic disease at diagnosis , the frequency of clinical symptoms and signs , the incidence of perianal disease , the incidence of extraintestinal manifestations , and the list of presumptive diagnosis on initial evaluation . and , we also compared the results of our patient population with representative experiences reported in the medical literatures .
the total number of the patients was only 45 , and of the 45 patients , 25 were males .
the male to female ratio was 1.3 to 1 with a slight preponderace to male .
1 ) ranged from 8 to 72 years , and the mean age was 35.5 .
the disease was relatively uncommon before the age of ten , and the peak incidence occurred in the 3rd , 4th and 5th decades and declined therafter .
nearly one third had the disease diagnosed before their 30th birthday , two thirds between their 20th and 50th birthday , and three fourths before their 50th birthday .
the patients with crohn s disease were divided into those with small bowel disease alone , those with ileocolitis , those with disease involving only the colon and the rectum .
nearly 60 per cent of the patients had macrosocpic disease confined to the ileum , and the ileum was involved in more than 75 per cent of the patients .
the colon was also involved in one third of the patients , and in only 20 per cent of the cases is the colon affected without concomitant small bowel disease , illustrating the diffuse nature of the disorder .
it appears that the vast majority of small and large bowel diseases mainly involve the ileum , cecum and ascending colon .
two patients had macroscopic diseases confined to the rectum , two to the sigmoid , and one to the descending colon .
table 2 shows representative experience reported in the medical literatures , and of our patient population .
the proportion of patients with small bowel , small and large bowel , and large bowel disease at diagnosis shows a significantly greater portion of patients with distal small bowel involvement compared with the patients studied by other authors .
there was a corresponding significant reduction in the proportion of patients with macroscopic colonic disease at diagnosis .
comparison of the anatomic distribution of lesions of crohn s disease in different centers is shown in table 3 .
it is worthy of notice that the anatomic distribution of lesions of crohn s disease in our patients is quite similar to that observed by dr .
the proportion of the patients with macroscopic disease confined to large bowel alone was only 15.6 per cent .
the most common symptom observed was abdominal pain , occurring in 89 per cent of the patients .
nonspecific symptoms such as anorexia , indigestion , weight loss were observed in one fourth of the patients .
table 5 shows the frequency of clinical signs according to the location of the disease .
abdominal mass was palpable in more than half the cases , which made it difficult to differentiate crohn s disease from cancer of colon , especially from the one with predominant infiltration of the bowel wall and secondary ulcer formation .
the frequency of perianal disease was also low in our patients , occurring in 11 per cent .
extraintestinal findings and their relationship to disease sites are shown in table 7 . in our series of 45 patients , only three patients presented with symptoms of arthritis .
other extraintestinal manifestations such as skin lesions , hepatobiliary manifestations , renal complications , eye complications were not observed at all .
table 8 shows the list of diagnosis which had been originally made on initial evaluation .
only 7 out of 45 patients had been presumed to have crohn s disease from the onset .
two fifths of the patients had been diagnosed colon cancer , because abdominal mass had been palpable in the majority of these patients .
ten patients had been initially thought to have intestinal tuberculosis , and two patients who had been presumed to have acute appendicitis turned out to have crohn s disease after surgical intervention .
it is now known that there are highly varying difference in the reported incidence of inflammatory bowel disease throughout the world and there are no areas which careful search has found to be free of these disorders . hahn and choi in our hospital reported 23 cases of crohn s disease .
a review of the literature shows another 22 cases were reported in korea by 1985 .
so the total number of the patients with crohn s disease that had been reported in korea during the period of 34 years from 1952 to 1985 only 45 .
it is not well understood why the disorder is so rare in korea , but there is a possibility that the true incidence of crohn s disease would have been masked because of confusion with intestinal tuberculosis and diarrhea of uncertain etiology which are quite common in korea .
table 9 is a list of illnesses that could potentially be considered in the differential diagnosis of crohn s disease in korea .
when , as is often the case , the disease begins abruptly with an apparantly transient episode of diarrhea , abdominal cramps and fever , the physician is likely to pass this initial episode off as salmonella infection which is quite endemic in korea . during the early , acute phase of the illness , crohn s disease
tuberculous involvement of bowel and peritoneum remains a major medical problem in korea , and it is extremely difficult to differentiate intestinal tuberoulosis from crohn s disease because the two disease share cardinal features , including diarrhea , abdominal pain , fever , weight loss , and radiologic demonstration of ileocecal involvement , with x - ray findings of intramural swelling , stricture and fistulas .
amebiasis , which is still common in korea , may be difficult to differentiate from crohn s disease , especially when localized to terminal ileum and cecum causing a chronic intramural lesion . because the abdominal mass is palpable in more than half the cases ,
it is difficult to make a differential diagnosis of crohn s disease from colon cancer , particularly if the latter lesion predominantly infiltrates the bowel wall and associated with secondary ulcer formation , which sometimes causes sinus tracts and fistulas into bowel or bladder .
the ratio of male to female patients was 1.3:1 ( 25 males and 20 females ) .
the age at diagnosis of the patients did not show a typical bimodal distribution which had been observed in other series .
a review of the literature shows that there has been considerable change in the anatomic distribution of crohn s disease over the past 50 years , and the proportion of patients with small bowel , small and large bowel , and large bowel disease at diagnosis shows recently a significantly greater proportion of patients with large bowel involvement at diagnosis .
there was a corresponding significant reduction in the proportion of patients with macroscopic ileal disease at diagnosis .
it is quite interesting to find the anatomic distribution of lesions of crohn s disease in our patients is similar to that observed by dr .
the literature shows that the symptoms of diarrhea is one of cardinal features of crohn s disease and is present in virtually all patients .
but , in our patients group , diarrhea was observed in only 13 per cent of the patients .
the exact reason why the symptom was rarely observed in the crohn s disease in korea is not well understood , but there is a possibility that crohn s disease presenting diarrhea as its chief clinical manifestation has been overlooked , because diarrhea of uncertain etiology is still prevalent in korea , masking the true incidence of crohn s disease .
an abdominal mass was palpable in more than half the cases , which made it difficult to differentiate crohn s disease from colon cancers .
over 40 per cent of the patients had been presumed to have colon cancer on initial evaluation , and that is one of the reasons why most cases in korea have been reported by surgeons .
intestinal obstruction was observed in 11 per cent of the patients , and the frequency of the lesion was quite lower than those reported by other authors .
the frequency of occurrence and detection of perianal disease is known to vary with their classification and the care of the examination , and it has also been shown that perianal disease is more common in crohn s disease and one half with crohn s colitis develop symptomatic perianal or perirectal fistulas .
the overall incidence of perianal disease in our patients was only 11 per cent , and it is suggested that low incidence of perianal disease might be due to small percentage of the disases with macroscopic involvement of large bowel . it is now known that systemic manifestations develop in crohn s disease , particularly in those individuals who have colonic involvement , about as often as they occur in ulcerative colitis .
extraintestinal manifestations also appear to occur more frequently among patients with perianal disease . in our series of 45 patients ,
only three patients presented symptoms of arthritis , and other extraintestinal manifestations such as skin lesions , hepatobiliary manifestations , renal complications , eye complications were not observed at all . in conclusion ,
crohn s disease is a quite rare disease among koreans , so it may be that this review on inflammatory bowel disease can not be given in statistically meaningful terms , and it would be better to accept the results of the data as a simple indication of the existence of crohn s disease among korean people . | crohn s disease is a rare disease in korea , and only 45 cases have been reported during a 34 year from 1952 to 1985.the male to female ratio was about 1.3 to 1 with a slight preponderance of males .
the age at diagnosis ranged from 8 to 72 ( mean 35.5 ) years , and the peak incidence occurred in the 3rd , 4th and 5th decades and declined thereafter.more than two thirds of the cases had a grossly demonstrable lesion involving the small bowel including the terminal ileum .
the proportion of the patients with macroscopic disease confined to the large bowel alone was only 15 per cent.abdominal pain was common presenting in 89 per cent of the patients , while such symptoms as fever , hematochezia and diarrhea were not common .
abdominal mass was palpable in more than half the cases , which made it difficult to differentiate crohn s disease from cancer of the colon , especially from the one with a predominant infiltration of the bowel wall and a secondary ulcer formation .
that is one of the reasons why most cases in korea have been reported by surgeons.a wide variety of complications were present , of which small bowel obstruction was the most common .
other complications were free perforation , malnutrition , fistula formation , hemorrhage and abscess formation in decreasing order.the incidence of symptomatic perianal diseas was only 11 per cent , and this might be due to the small proportion of the disesase confined to large bowel.extraintestinal manifestations were also rare , and only three patients presented the symptoms of arthritis .
other systemic features such as liver disease , skin lesions , eye complicaitons were absent . |
both registry data1 , 2 , 3 , 4 and secondary analyses of randomized controlled trials5 , 6 , 7 have suggested that the burden of anemia in patients presenting with acute coronary syndromes ( acs ) is significant .
a recent metaanalysis of 27 studies including 233 144 patients has reported a prevalence of anemia in acs patients close to 20%,8 and current clinical guidelines fail to offer firm recommendations for its concurrent management in the acs setting.9 , 10
patients with anemia are older3 , 6 , 7 , 11 with a significantly greater burden of comorbidities such as chronic kidney disease,12 , 13 , 14 diabetes,6 , 13 , 14 heart failure,13 , 15 and more extensive coronary artery disease6 and are less likely to undergo cardiac catheterization.4 , 6 , 11 these adverse clinical characteristics are well known to contribute to adverse outcomes in patients with acs .
previous reports have suggested that acs patients with anemia have significantly worse inhospital and longerterm total and cardiac mortality outcomes,5 , 6 , 7 , 16 , 17 heart failure,18 and risk of major bleeding6 and of reinfarction.6 , 8 , 19 some studies have reported that , once differences in age or comorbidity burden between anemic / nonanemic acs cohorts are adjusted for , anemia is no longer an independent predictor of adverse mortality20 or cardiovascular mortality,21 although other studies report that the relationship persists.6 , 7 , 19 , 22 other studies have reported different relationships between anemia and cardiovascular ( cv ) outcomes according to sex , with baseline anemia independently associated with higher rates of allcause and cardiac mortality at 30 days and 1 year in men but not in women.7
data derived from secondary analyses from randomized controlled trials ( rct ) have suggested adverse mortality outcomes associated with anemia in patients with acs,5 , 6 , 23 but such rcts often exclude older patients with the most severe comorbid conditions and may therefore underreport the prevalence of anemia and underestimate its prognostic impact .
many of the studies that have reported relationships between anemia and adverse outcomes in the setting of acs have not adjusted for or excluded patients with major bleeding events3 , 5 , 24 that may further confound the relationships reported .
we have estimated the prevalence of anemia in acs patients and compared their clinical characteristics , management , and clinical outcomes to those without anemia in an unselected national acs cohort derived from the myocardial ischaemia national audit project ( minap ) registry , which collects data on all patients in the uk admitted with a confirmed diagnosis of acs .
we also examined the relationship between anemia and short ( 30day ) and longerterm ( 1year ) mortality outcomes in this setting and assessed whether the prognostic impact of anemia relates to its severity .
the minap registry collects data on all patients aged 18 or over in the united kingdom who are admitted to all 230 nhs hospital trusts in england and wales with a confirmed diagnosis of an acs .
we analyzed data from the registry for patients admitted between january 2006 and december 2010 on this registry and followed them up until august 2011 .
participants were included in the current study if they had a diagnosis of any acs ( stsegment elevation myocardial infarction [ stemi ] , nonstsegment elevation myocardial infarction [ nstemi ] , or unstable angina ) determined by the medical team at time of discharge .
mortality outcome was ascertained by linkage through the office of national statistics.25
the minap data set collects standardized data on prehospital and inhospital care for all acs admissions from all 230 nhs trusts in england and wales and is part of the nhs data dictionary ( http://www.hqip.org.uk/minap-2013-report/ ) .
the details of development and initial findings are reported elsewhere.26
in the current study variables included in the analyses were hemoglobin at the time of admission with an acs , age , sex , smoking status , peak troponin levels , hyperlipidemia , hypertension , prior angina , prior myocardial infarction , prior heart failure , prior stroke , peripheral vascular disease , chronic obstructive pulmonary disease , diabetes , renal failure , prior percutaneous coronary intervention , prior coronary artery bypass graft , prior medications ( angiotensinconverting enzyme [ ace ] inhibitor , blocker , statin , clopidogrel , aspirin ) , clinical diagnosis ( unstable angina , nstemi , stemi ) , discharge medications ( ace inhibitor , blocker , statin , clopidogrel , aspirin ) , angiography , inhospital bleeding , and mortality outcomes in hospital and within 30 days and 1 year .
world health organization ( who ) hemoglobin thresholds were used to define anemia as < 13 g / dl for men and < 12 g / dl for women .
multiple imputations by chained equations in stata version 13.0 were used to impute missing values for variables where possible .
we describe baseline variables according to whether they were missing or nonmissing in tables , and details of participant inclusion are shown graphically .
descriptive statistics are presented for baseline variables and outcome according to anemia status and sex .
associations between anemia status and individual variables were tested using 1way analysis of variance for continuous variables and chisquared test for categorical variables .
we used multiple logistic regression with adjustments for baseline variables to determine factors associated with anemia .
the same regression methods were used to determine the adjusted odds of 30day mortality with 1 g / dl incremental hemoglobin increase from < 10 to 18 g / dl for men and from < 9 to 17 g / dl for women .
additional analyses were performed to determine the adjusted odds ratio of mortality at 30 days and 1 year for anemic compared to nonanemic groups for the whole cohort , the maleonly cohort , femaleonly cohort , nstemi cohort , stemi cohort , and subgroups where bleeding was excluded .
the baseline variables of the group of participants who bled and did not bleed were also compared .
severity of anemia was examined by stratifying the cohort by sexspecific hemoglobin cutoffs ( hb < 10 g / dl , hb 1011 g / dl , hb 1112
g / dl for men and hb < 9 g / dl , hb 910
g / dl for women ) . to better control for baseline differences across the anemic and nonanemic groups ,
further analysis was performed using propensity score matching ( mi estimate : teffects psmatch ) to estimate average treatment effects ( ate ) .
although multiple regression is the most widely used method to control for measured confounders , it can be inadequate when the 2 comparator groups ( anemia vs no anemia in our analyses ) are very different across key confounders .
propensity score matching can be a better approach in such extreme scenarios and can thus serve as a useful sensitivity analysis .
propensity scores were calculated using multiple logistic regression , and then 1:1 matching with replacement ( ie , including all cases and controls ) was performed prior to simple logistic regression models to obtain the ate .
the current study obtained the ethical approval from the faculty of medicine & health sciences research ethics committee , university of east anglia .
informed consent from participants was waived as data were routinely collected , and only anonymized data were used in the study .
the minap registry collects data on all patients aged 18 or over in the united kingdom who are admitted to all 230 nhs hospital trusts in england and wales with a confirmed diagnosis of an acs .
we analyzed data from the registry for patients admitted between january 2006 and december 2010 on this registry and followed them up until august 2011 .
participants were included in the current study if they had a diagnosis of any acs ( stsegment elevation myocardial infarction [ stemi ] , nonstsegment elevation myocardial infarction [ nstemi ] , or unstable angina ) determined by the medical team at time of discharge .
the minap data set collects standardized data on prehospital and inhospital care for all acs admissions from all 230 nhs trusts in england and wales and is part of the nhs data dictionary ( http://www.hqip.org.uk/minap-2013-report/ ) .
the details of development and initial findings are reported elsewhere.26
in the current study variables included in the analyses were hemoglobin at the time of admission with an acs , age , sex , smoking status , peak troponin levels , hyperlipidemia , hypertension , prior angina , prior myocardial infarction , prior heart failure , prior stroke , peripheral vascular disease , chronic obstructive pulmonary disease , diabetes , renal failure , prior percutaneous coronary intervention , prior coronary artery bypass graft , prior medications ( angiotensinconverting enzyme [ ace ] inhibitor , blocker , statin , clopidogrel , aspirin ) , clinical diagnosis ( unstable angina , nstemi , stemi ) , discharge medications ( ace inhibitor , blocker , statin , clopidogrel , aspirin ) , angiography , inhospital bleeding , and mortality outcomes in hospital and within 30 days and 1 year .
world health organization ( who ) hemoglobin thresholds were used to define anemia as < 13 g / dl for men and < 12 g / dl for women .
multiple imputations by chained equations in stata version 13.0 were used to impute missing values for variables where possible .
we describe baseline variables according to whether they were missing or nonmissing in tables , and details of participant inclusion are shown graphically .
descriptive statistics are presented for baseline variables and outcome according to anemia status and sex .
associations between anemia status and individual variables were tested using 1way analysis of variance for continuous variables and chisquared test for categorical variables .
we used multiple logistic regression with adjustments for baseline variables to determine factors associated with anemia .
the same regression methods were used to determine the adjusted odds of 30day mortality with 1 g / dl incremental hemoglobin increase from < 10 to 18 g / dl for men and from < 9 to 17 g / dl for women .
additional analyses were performed to determine the adjusted odds ratio of mortality at 30 days and 1 year for anemic compared to nonanemic groups for the whole cohort , the maleonly cohort , femaleonly cohort , nstemi cohort , stemi cohort , and subgroups where bleeding was excluded .
the baseline variables of the group of participants who bled and did not bleed were also compared .
severity of anemia was examined by stratifying the cohort by sexspecific hemoglobin cutoffs ( hb < 10 g / dl , hb 1011 g / dl , hb 1112 g / dl , hb 1213
g / dl for men and hb < 9 g / dl , hb 910
g / dl for women ) . to better control for baseline differences across the anemic and nonanemic groups ,
further analysis was performed using propensity score matching ( mi estimate : teffects psmatch ) to estimate average treatment effects ( ate ) .
although multiple regression is the most widely used method to control for measured confounders , it can be inadequate when the 2 comparator groups ( anemia vs no anemia in our analyses ) are very different across key confounders .
propensity score matching can be a better approach in such extreme scenarios and can thus serve as a useful sensitivity analysis .
propensity scores were calculated using multiple logistic regression , and then 1:1 matching with replacement ( ie , including all cases and controls ) was performed prior to simple logistic regression models to obtain the ate .
the current study obtained the ethical approval from the faculty of medicine & health sciences research ethics committee , university of east anglia .
informed consent from participants was waived as data were routinely collected , and only anonymized data were used in the study .
there were a total of 424 848 participants in the minap cohort between january 2006 and december 2010 who were followed up until august 2011 .
figure s1 shows the flow diagram of participant inclusion , and comparison of characteristics between those with and without available data on hb did not show any material differences ( table s1 ) .
the prevalence of anemia in this cohort was 71 223/256 744 ( 27.7% ) .
after multiple imputations the sample size of the complete data set with all imputed variables was 256 744 .
the descriptive statistics of baseline variables in the included cohort , sorted by anemia status , are shown in table 1 .
the anemic cohort was significantly older , with a higher proportion of smokers ( 85% vs 68% , p<0.001 ) , prior hypertension ( 59% vs 48% , p<0.001 ) , angina ( 43% vs 27% , p<0.001 ) , myocardial infarction ( 38% vs 23% , p<0.001 ) , prior heart failure ( 12% vs 4% , p<0.001 ) , stroke ( 14% vs 7% , p<0.001 ) , peripheral vascular disease ( 8% vs 3% , p<0.001 ) , copd ( 18% vs 14% , p<0.001 ) , diabetes ( 31% vs 16% , p<0.001 ) , and renal failure ( 19% vs 4% , p<0.001 ) .
participants who were anemic were more likely to have aspirin and clopidogrel prior to admission ( 7% vs 5% , p<0.001 ) and less likely to be prescribed dual antiplatelet therapy on discharge ( 75% vs 79% , p<0.001 ) .
participants whose anemia occurred in the context of a bleeding complication were more likely to be female , to be on aspirin before admission and on discharge ( 30% vs 28% , p<0.001 ) , to have stemi diagnosis ( 52% vs 38% , p<0.001 ) , to be less likely to receive angiography ( 26% vs 38% , p<0.001 ) and more likely to die at 30 days ( 6% versus 3% , p<0.001 ) and 1 year ( 12% vs 8% , p<0.001 ) ( table s2 ) .
similar rates of angiography were performed in the anemic versus nonanemic cohort , but patients with anemia were significantly less likely to be prescribed secondary prevention medications postdischarge .
the difference in crude mortality rates between the anemic and nonanemic groups increased with longer followup .
baseline characteristic of the minap cohort according to anemia status bmi indicates body mass index ; cabg , coronary artery bypass graft ; copd , chronic obstructive pulmonary disease ; minap , myocardial ischemia national audit project ; nstemi , nonstsegment elevation myocardial infarction ; pci , percutaneous coronary intervention ; pvd , peripheral vascular disease ; stemi , stsegment elevation myocardial infarction .
results reported as mean ( sd ) for continuous variables and n ( % ) for categorical variables . logistic regression ( continuous variables ) ,
multiple logistic regression was used to determine the independent factors associated with the presence of anemia at baseline ( table 2 ) .
the most significant associations were observed with presence of peripheral vascular disease ( or 1.427 , 95% ci 1.3621.496 , p<0.001 ) , diabetes mellitus ( or 1.786 , 95% ci 1.7421.832 , p<0.001 ) , and renal disease ( or 3.058 , 95% ci 2.9623.158 , p<0.001 ) .
significant factors associated with anemia ( n=422 855 ) : logistic regression modela
cabg indicates coronary artery bypass graft ; copd , chronic obstructive pulmonary disease ; pci , percutaneous coronary intervention ; pvd , peripheral vascular disease .
the adjusted odds of mortality by incremental ( 1 g / dl ) increase in hemoglobin are shown in figure 1 .
lower hemoglobin values were associated with significantly higher mortality with a nonsignificant trend toward higher mortality in those patients with elevated hemoglobin values . adjusted odds of mortality at 30 days according to hemoglobin levels for men and women . adjusted for age , sex ,
current or exsmokers , troponin , hyperlipidemia , hypertension , prior angina , prior myocardial infarction , prior heart failure , stroke , peripheral vascular disease , chronic obstructive pulmonary disease , diabetes , renal failure , prior percutaneous coronary intervention , prior coronary artery bypass graft , medications prior to admission , diagnosis , medications at discharge and angiography .
the odds of mortality associated with the presence of anemia following adjustment for baseline covariates are shown in table 3 .
we observed that there was a ~1.3fold increase in odds of 30day mortality ( or 1.281 , 95% ci 1.2171.350 , p<0.001 ) and 1year ( or 1.311 , 95% ci 1.2741.348 , p<0.001 ) mortality , respectively , with anemia after adjustment for potential confounders .
similar significant increases in mortality with anemia were observed for men ( 30day mortality or 1.298 , 95% ci 1.2171.384 , p<0.001 ; 1year mortality or 1.354 , 95% ci 1.2991.411 , p<0.001 ) and women ( 30day mortality or 1.255 , 95% ci 1.1461.374 , p<0.001 ; 1year mortality or 1.252 , 95% ci 1.1981.309 , p<0.001 ) as well as diagnosis of nstemi ( 30day mortality or 1.291 , 95% ci 1.2131.374 , p<0.001 ; 1year mortality or 1.326 , 95% ci 1.2811.374 , p<0.001 ) and stemi ( 30day mortality or 1.269 , 95% ci 1.1631.384 , p<0.001 ; 1year mortality or 1.284 , 95% ci 1.2841.352 , p<0.001 ) . in order to eliminate the potential confounding influence of major bleeding complications on the relationship between anemia and mortality , we repeated the analysis following exclusion of patients with major bleeding complications , and similar results were recorded ( 30day mortality or 1.279 , 95% ci 1.2131.348 , p<0.001 ; 1year mortality or 1.309 , 95% ci 1.2711.347 , p<0.001 ) .
furthermore , a sensitivity analysis was undertaken in patients in whom hb values were recorded at baseline ( 257 999 patients ) , and similar independent factors associated with anemia ( table s3 ) and mortality outcomes associated with anemia ( table s4 ) were observed .
multivariate association between anemia and mortality : logistic regression models nstemi indicates nonstsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction .
propensity score matched analysis is shown in table 4 , and anemia is associated with significant increase in mortality at both 30 days and 1 year after adjustments for propensity score ( 30day mortality coefficient 0.0080 , 95% ci 0.00450.0114 , p<0.001 ; 1year mortality coefficient 0.0173 , 95% ci 0.01280.0218 , p<0.001 ) .
propensity score matching analysis on 10 imputed data sets , reporting average treatment effects ( ate ) there were also differences in baseline characteristics according to sex ( table 5 ) .
most notably , females were younger ( mean age 74 vs 67 years , p<0.001 ) , but more were smokers ( 79% vs 70% , p<0.001 ) and hypertensive ( 56% vs 47% , p<0.001 ) . however , medication at discharge was higher in men , and women had a higher proportion of patients with adverse outcomes .
baseline characteristic of the minap cohort in a single imputed data set according to sex bmi indicates body mass index ; cabg , coronary artery bypass graft ; copd , chronic obstructive pulmonary disease ; minap , myocardial ischemia national audit project ; nstemi , nonstsegment elevation myocardial infarction ; pci , percutaneous coronary intervention ; pvd , peripheral vascular disease ; stemi , stsegment elevation myocardial infarction .
results reported as mean ( sd ) for continuous variables and n ( % ) for categorical variables . logistic regression ( continuous variables ) ,
the sexspecific adjusted odds of mortality by incremental ( 1 g / dl ) increase in hemoglobin is shown in figure 2 .
for both sexes , lower hemoglobin values were associated with significantly higher mortality , but high values of hemoglobin were associated with higher mortality only in men and not in women .
similar results were observed if patients with bleeding were excluded ( figure s2 ) . adjusted odds of mortality at 30 days according to hemoglobin levels and sex .
adjusted for age , current or exsmokers , troponin , hyperlipidemia , hypertension , prior angina , prior myocardial infarction , prior heart failure , stroke , peripheral vascular disease , chronic obstructive pulmonary disease , diabetes , renal failure , prior percutaneous coronary intervention , prior coronary artery bypass graft , medications prior to admission , diagnosis , medications at discharge , and angiography . in terms of severity of anemia
there was an increase in mortality at both 30 days and 1 year with reduced hemoglobin , which ranged from ~1.2 to 1.3fold increase in odds of mortality for hb 12 to 13 g / dl to a ~1.4 to 1.5fold increase for hb < 10 g / dl for men ( table 6 ) .
our analysis is the largest analysis to study the prevalence , clinical characteristics , and outcomes associated with anemia in an unselected national cohort of acs patients in the united kingdom .
we have observed that more than 1 in 4 patients presenting with acs are anemic and that these patients are older , have a greater prevalence of comorbid conditions , and are less likely to receive evidencebased therapies shown to improve clinical outcomes .
finally , our analysis suggests that anemia is independently associated with adverse inhospital and longerterm mortality outcomes , with a reverse jshaped relationship between hb levels and both short and longer mortality outcomes observed .
our observed prevalence of anemia of 28% is greater than that reported in data derived from rcts reporting rates of between 10% and 25%,5 , 6 , 7 , 23 although registry data reveal significantly higher prevalences.4 , 12 , 27 for example , an analysis of 78 974 medicare beneficiaries aged 65 years or older hospitalized with acute myocardial infarction revealed a prevalence of anemia of 43%.12 we have observed that acs patients with anemia are older , have a greater prevalence of comorbid conditions , and are less likely to receive evidencebased therapies for the treatment of acs , in agreement with previous literature.5 , 6 , 7 , 19 , 21
in the current analysis we report that the presence of anemia is independently associated with an ~50% increased risk of mortality in the short and long term and that this prognostic impact is observed in both men and women , in contrast to the findings of a secondary analysis of the horizonsami trial that failed to demonstrate an association between anemia and increased risk of mortality in women.7 we have observed a reverse jshaped relationship between decreasing hb values and 30day mortality , with a doseresponse effect with progressively lower odds of survival with more profound degrees of anemia in both men and women .
similar reverse jshaped relationships are seen between hb levels and cv death and the composite endpoint of cv death , myocardial infarction , or recurrent ischemic events in some studies,21 although other studies have not revealed such statistically significant relationships in either inhospital cardiac mortality16 or longerterm mortality.1
previous studies have reported that anemia is independently associated with adverse clinical outcomes , but many of these studies did not report whether patients with bleeding events were excluded from their analyses , as it is well documented that major bleeding is independently associated with mortality in the acs setting,28 , 29 , 30 which might have confounded any reported relationships between the presence of anemia and mortality . in the current analysis
we report anemia independently predicts adverse mortality outcomes and that the jshaped relationship between hb level and mortality persists even after exclusion of patients with bleeding events .
there are several biological and clinical reasons why anemia may lead to worse clinical outcomes in patients with acs . in the setting of acs
, anemia might worsen ischemia by decreasing the oxygen delivery to the jeopardized myocardium and increase myocardial oxygen demand due to greater cardiac output to maintain adequate systemic oxygen delivery.31 , 32 clinically , patients with anemia are often underprescribed antiplatelet therapy due to bleeding concerns ; for example , in our current analysis clopidogrel was prescribed in 73% of patients without anemia and 66% with anemia ( p<0.001 ) , whereas in the cadillac trial 18% of patients with anemia at the time of their acs were no longer receiving aspirin at 1 year,23 which might contribute to increased cardiovascular events .
analysis of the acuity trial suggests that patients with anemia were less likely to undergo percutaneous coronary intervention ( pci ) and more likely to be medically managed , which may further contribute to worse cardiovascular outcomes in this group.6 finally , anemia may be a manifestation of numerous chronic disease states , and the presence of anemia is merely a marker of poorer outcomes in patients with chronic diseases .
our analysis suggests that anemia is independently associated with adverse clinical outcomes in patients presenting with acs .
there is a lack of clarity in contemporary guideline recommendations as to whether such patients with anemia should be transfused and the optimal transfusion strategy.33 the american association of blood banks recommendation for patients presenting with acs is no recommendation for or against a liberal or restrictive transfusion threshold for hospitalized , hemodynamically stable patients with acute coronary syndrome.33 furthermore , the crit ( conservative vs liberal red cell transfusion in acute myocardial infarction ) randomized pilot trial34 demonstrated that patients with acs and a hematocrit level < 30% who were randomized to a liberal transfusion arm had a significantly higher composite endpoint of inhospital death , recurrent myocardial infarction , or congestive heart failure than those who underwent more restrictive transfusion practice ( 38% vs 13% ; p=0.046 ) .
in contrast , in the mint ( a multicenter , randomized study of argatroban versus heparin as adjunct to tissue plasminogen activator [ tpa ] in acute myocardial infarction : myocardial infarction with novastan and tpa ) pilot study undertaken in 110 patients presenting with an acs or stable angina with anemia undergoing cardiac catheterization , patients randomized to a liberal blood transfusion strategy had 50% lower primary outcome rates of death , myocardial infarction , and unscheduled revascularization compared to those patients randomized to a restrictive transfusion strategy , with lower 30day mortality too .
a recent metaanalysis of 10 studies consisting mainly of registry studies including 203 665 patients has shown a close to 3fold independent increase in the risk of mortality associated with a liberal blood transfusion strategy in the ami setting with metaregression adjusting for a history of bleeding or baseline hemoglobin level revealing a similar increased risk , indicating a significant risk for blood transfusion over and above that associated with bleeding or anemia,35 with similar findings reported in the pci setting.36
our study has some limitations .
the minap data set requires the recording of hb within 24 hours of admission .
many of these hb values , particularly in the setting of hemodynamically unstable nstemi or stemi treated with primary pci , may be postpci and may reflect the influence of acute bleeding complications and not reflect chronic anemia . nevertheless , even following exclusion of patients who sustained bleeding complications during their inhospital course , the relationships that we examined remained unchanged .
we report an association between anemia and inhospital and longerterm mortality , but we can not infer causality . although it would be interesting to know whether anemia was associated with cardiac mortality , we were unable to determine the cause of death for participants .
we have adjusted for differences in baseline characteristics between the anemic and nonanemic cohorts , but other unmeasured confounders may be contributing to the adverse clinical outcomes associated with anemia that we report .
another limitation was the missing data , which varied in extent depending on the study variable , and we tried to approximate these values using multiple imputations to impute the missing values .
finally , the minap data set does not record the receipt of blood transfusions , which may contribute to the adverse clinical outcomes reported.35
in conclusion , this is the largest study of the prevalence , clinical characteristics , and outcomes associated with anemia in an unselected national cohort of acs patients in the united kingdom .
we report a significant prevalence of anemia in a contemporary acs cohort , with approximately 1 in 4 patients presenting with acs being anemic , and that these patients are older , have a greater prevalence of comorbid conditions , and are less likely to receive evidencebased therapies shown to improve clinical outcomes .
finally , our findings suggest that anemia is independently associated with adverse 30day and longerterm mortality outcomes , with a reverse jshaped relationship between hb levels and mortality outcomes observed in both men and women .
the clinical effectiveness of correcting anemia routinely in acs has not been widely explored , and there is considerable uncertainty in the value of such an approach .
mamas , zaman , and myint conceived and planned the study . kwok and kontopantelis analyzed the data . mamas and
myint is the guarantor . myint and zaman are copis of the minapolder age project .
the project was funded by the halley stewart trust and the north staffordshire medical institute .
mrc health eresearch centre grant mr / k006665/1 supported the time and facilities of kontopantelis .
this work was undertaken by the research team and funded by the sir halley stewart trust , medical research council , and north staffordshire medical institute .
the views expressed within this paper are those of the authors and not necessarily those of the funders .
table s1 . comparison of patients with missing and no missing hemoglobin values
table s2 .
significant multivariate predictors of anemia in patients in whom baseline hemoglobin values were recorded ( n=256 744 )
table s4 .
sensitivity analysis of multivariate association between anemia and mortality in patients in whom baseline hemoglobin values were recorded
figure s1 .
figure s2 . adjusted odds of mortality at 30 days according to hemoglobin levels and sex with exclusion of participants with bleed outcome . | backgroundwe aim to determine the prevalence of anemia in acute coronary syndrome ( acs ) patients and compare their clinical characteristics , management , and clinical outcomes to those without anemia in an unselected national acs cohort.methods and resultsthe myocardial ischemia national audit project ( minap ) registry collects data on all adults admitted to hospital trusts in england and wales with diagnosis of an acs .
we conducted a retrospective cohort study by analyzing patients in this registry between january 2006 and december 2010 and followed them up until august 2011 .
multiple logistic regressions were used to determine factors associated with anemia and the adjusted odds of 30day mortality with 1 g / dl incremental hemoglobin increase and the 30day and 1year mortality for anemic compared to nonanemic groups .
analyses were adjusted for covariates .
our analysis of 422 855 patients with acs showed that 27.7% of patients presenting with acs are anemic and that these patients are older , have a greater prevalence of renal disease , peripheral vascular disease , diabetes mellitus , and previous acute myocardial infarction , and are less likely to receive evidencebased therapies shown to improve clinical outcomes .
finally , our analysis suggests that anemia is independently associated with 30day ( or 1.28 , 95% ci 1.221.35 ) and 1year mortality ( or 1.31 , 95% ci 1.271.35 ) , and we observed a reverse jshaped relationship between hemoglobin levels and mortality outcomes.conclusionsthe prevalence of anemia in a contemporary national acs cohort is clinically significant .
patients with anemia are older and multimorbid and less likely to receive evidencebased therapies shown to improve clinical outcomes , with the presence of anemia independently associated with mortality outcomes . |
with the advent of newer imaging modalities and their widespread availability , brain arteriovenous malformations ( avms ) are being increasingly diagnosed . even though microsurgery is advocated as the definitive treatment modality , radiosurgery is commonly performed , as an alternative to surgical resection in many centers .
the avm situated in and around the eloquent regions of the brain and those in the deeper regions of the brain are favored for radiosurgery .
the principle of stereotactic radiosurgery is to accurately deliver a high dose of precise conformal radiation to an imaging - defined target in a single fraction .
nonetheless , the tissue adjacent to a radiosurgical target does receive a dose of radiation , albeit being a very small fraction of the total dose .
given the fact that majority of the patients with avm are young , with long years of productive life ahead , the concern of radiation injury to the brain and its attendant adverse effects on cognitive function arise .
very few studies have addressed the issue of the possibility of cognitive dysfunction in patients who undergo radiosurgery .
the present study evaluated a cohort of patients with brain avms prospectively and compared the pretherapeutic neuropsychological scores with 2 years posttreatment scores . to the best of our knowledge
, this study represents one of the largest series where cognitive outcome after 2 years of patients with avms treated with gamma knife radiosurgery ( gkrs ) has been evaluated .
this prospective study included all the patients who underwent gkrs at our institute for avm of the brain between 2006 and december 2008 .
inclusion criteria included the age range of 1655 years . only patients who were alert and amenable for neuropsychological evaluation
patients who had also undergone other modalities of treatment like embolization or surgery prior to gkrs were excluded . in the pretreatment period ,
a repeat neuropsychological evaluation 2 years following gkrs could be performed on 18 patients by the same examiner .
we present in detail the comparative data of these 18 patients who were evaluated before gkrs and at 2 years follow - up period .
patient characteristics and treatment details neuropsychological evaluation was performed using nimhans neuropsychology battery , with various tests as outlined in table 2 .
all the tests were administered by a single examiner . the session for a patient ranged about 90180 min for the completion of all tests .
neuropsychological tests finger tapping tests and digit symbol substitution tests were used to assess motor speed and mental speed , respectively .
tests of attention included color trails test , digit vigilance test , and triads test .
tests of executive functions included controlled oral word association test , animal names test , design fluency test , n - back tests ( verbal and visual ) , the tower of london test , wisconsin card sorting test ( wcst ) , and stroop test .
wcst details concept formation and set shifting ability while stroop test was used as an index of response inhibition .
auditory verbal learning test and rey 's complex figure test were used to assess verbal and visual learning and memory respectively .
radiosurgery was performed with leksell gamma knife 4c system . in brief , patients underwent magnetic resonance imaging ( mri ) of the brain and biplanar digital subtraction angiography ( dsa ) after fixation of leksell stereotactic frame .
images were imported to gkrs planning system . a conformal gkrs plan with the prescription isodose , maximum dose , and dose to the margin was determined [ figure 1 g ] .
follow - up consisted of a 6 monthly neurological examination and mri of the brain .
obliteration is defined as the complete absence of pathological vessels in the former nidus of the avm , and the disappearance or normalization of afferent and efferent pathological vessels and a normal circulation time .
subtotal obliteration implies complete angiographic disappearance of the nidus , but the persistence of an early draining vein , as defined previously . ( a and b ) left internal carotid angiogram demonstrating an arteriovenous malformation in the left splenial region being fed by the distal anterior cerebral artery and the posterior choroidal branches of the posterior cerebral artery .
( c and d ) follow - up digital subtraction angiography after gamma knife radiosurgery shows complete obliteration of the arteriovenous malformation .
( e ) magnetic resonance imaging brain t2 axial sequences show an arteriovenous malformation in the left precentral gyrus .
( f ) magnetic resonance imaging brain t2 axial sequences following gamma knife radiosurgery showed partial obliteration of arteriovenous malformation with extensive hyperintensities suggestive of postradiosurgery imaging changes .
( g ) a screen shot of the gamma plan of a corpus callosal arteriovenous malformation being treated with gamma knife radiosurgery neuropsychological improvement at 2 years follow - up ; comparison between pretreatment and at 2 years follow - up scores initial data obtained after pretreatment evaluation were screened to look for general cognitive deficits seen in this patient group .
the 2 years posttreatment test evaluation was compared with pretreatment data using wilcoxon signed ranks test .
all statistical analysis was performed using statistical package for social sciences v. 15 ( spss inc . ,
this prospective study included all the patients who underwent gkrs at our institute for avm of the brain between 2006 and december 2008 .
inclusion criteria included the age range of 1655 years . only patients who were alert and amenable for neuropsychological evaluation
patients who had also undergone other modalities of treatment like embolization or surgery prior to gkrs were excluded . in the pretreatment period ,
a repeat neuropsychological evaluation 2 years following gkrs could be performed on 18 patients by the same examiner .
we present in detail the comparative data of these 18 patients who were evaluated before gkrs and at 2 years follow - up period .
neuropsychological evaluation was performed using nimhans neuropsychology battery , with various tests as outlined in table 2 .
all the tests were administered by a single examiner . the session for a patient ranged about 90180 min for the completion of all tests .
neuropsychological tests finger tapping tests and digit symbol substitution tests were used to assess motor speed and mental speed , respectively .
tests of attention included color trails test , digit vigilance test , and triads test .
tests of executive functions included controlled oral word association test , animal names test , design fluency test , n - back tests ( verbal and visual ) , the tower of london test , wisconsin card sorting test ( wcst ) , and stroop test .
wcst details concept formation and set shifting ability while stroop test was used as an index of response inhibition .
auditory verbal learning test and rey 's complex figure test were used to assess verbal and visual learning and memory respectively .
radiosurgery was performed with leksell gamma knife 4c system . in brief , patients underwent magnetic resonance imaging ( mri ) of the brain and biplanar digital subtraction angiography ( dsa ) after fixation of leksell stereotactic frame .
images were imported to gkrs planning system . a conformal gkrs plan with the prescription isodose , maximum dose , and dose to the margin was determined [ figure 1 g ] .
follow - up consisted of a 6 monthly neurological examination and mri of the brain .
obliteration is defined as the complete absence of pathological vessels in the former nidus of the avm , and the disappearance or normalization of afferent and efferent pathological vessels and a normal circulation time .
subtotal obliteration implies complete angiographic disappearance of the nidus , but the persistence of an early draining vein , as defined previously .
( a and b ) left internal carotid angiogram demonstrating an arteriovenous malformation in the left splenial region being fed by the distal anterior cerebral artery and the posterior choroidal branches of the posterior cerebral artery .
( c and d ) follow - up digital subtraction angiography after gamma knife radiosurgery shows complete obliteration of the arteriovenous malformation .
( e ) magnetic resonance imaging brain t2 axial sequences show an arteriovenous malformation in the left precentral gyrus .
( f ) magnetic resonance imaging brain t2 axial sequences following gamma knife radiosurgery showed partial obliteration of arteriovenous malformation with extensive hyperintensities suggestive of postradiosurgery imaging changes .
( g ) a screen shot of the gamma plan of a corpus callosal arteriovenous malformation being treated with gamma knife radiosurgery neuropsychological improvement at 2 years follow - up ; comparison between pretreatment and at 2 years follow - up scores
initial data obtained after pretreatment evaluation were screened to look for general cognitive deficits seen in this patient group . the 2 years posttreatment test evaluation was compared with pretreatment data using wilcoxon signed ranks test .
all statistical analysis was performed using statistical package for social sciences v. 15 ( spss inc . ,
the present study noted that , out of the total patient cohort with avm ( n = 34 ) , more than half had a significant deviation of neuropsychological functions from the normal population norms .
the functions of attention , verbal learning , and memory and visual working memory were commonly affected .
the present study noted that impairment in motor speed in 26.5% of patients , mental speed in 28.6% , focused attention in 36.7% , sustained attention in 58.3% , verbal fluency in 29.4% , response inhibition in 24.4% , visual working memory in 100% , and verbal working memory in 37.5% of patients . in verbal learning and memory , deficits were noted for verbal encoding in 56.2% , verbal retrieval in 56.2% , and verbal recognition in 72% .
three other patients had subtotal obliteration . based on the criteria defined by pollock and flickinger 12 patients ( 66.6% ) achieved an excellent outcome , i.e. obliteration of nidus and no new deficits [ figure 1a - d ] .
the outcome was fair in one patient ( obliteration and a major deficit ) . in three patients with subtotal
one other patient , who is a housewife who had transient hemiparesis , could resume light household activities .
the neuropsychological outcome was compared in those 18 patients who had evaluation both preprocedure and at the 2 years follow - up .
, patients showed an improvement in neuropsychological function at various categories . among them , the prominent statistically significant improvement was noted for set - shifting ability .
pretherapeutic median value for percentage perseverative responses was 26.5 and at follow - up , it reduced to 18.2 ( p = 0.039 ) .
set shifting improved in 11 patients ( 61.1% ) , remained same in 5 patients ( 27.7% ) , and deteriorated in two patients ( 11.1% ) .
other notable improvement was an increase in efficiency of planning in reduced time ( tested by the tower of london test ) .
patients took a median time of only 7.5 s as opposed to 16 s for successfully completing the 2 moves a section of the test ( p = 0.044 ) .
patients also showed improved ability for response inhibition at follow - up ( p = 0.003 ) .
the factors which are likely to influence the neuropsychological improvement namely volume of avm , location of the avm in the brain , spetzler - martin grade , radiosurgery based avm score , and marginal dose were evaluated for their impact on the outcome .
the effect of obliteration could not be analyzed because all avms showed some response at 2 years follow - up ( 15 patients showed total obliteration , and three had subtotal obliteration ) . similarly , the presence of intracranial hematoma also could not be analyzed as only two out of 18 patients had nonhemorrhagic presentation . on analysis ,
interestingly , patients with a higher spetzler - martin grade avm demonstrated a significantly more favorable shift in follow - up test values for set shifting function ( p = 0.021 ) .
post radiosurgery imaging changes have been defined as mild , moderate , and severe in previous studies .
five patients in the present series revealed imaging changes at various stages of follow - up [ figure 1e and f ] .
three patients had mild changes ; one had moderate pri , and one had severe pri . the mean duration for the appearance of pri after treatment was 10.8 5 months .
on assessing individual patient 's performance in relation to pri severity , it was noted that patients with postradiation imaging changes had a lesser tendency to improve in neuropsychological performance in follow - up tests .
four out of five patients with pri changes demonstrated some neuropyschological deterioration at follow - up evaluation .
one patient with moderate pri and two patients with mild pri showed deteriorated conceptual responses at follow - up .
one patient with severe pri showed deteriorated set shifting function on follow - up evaluation .
the present study noted that , out of the total patient cohort with avm ( n = 34 ) , more than half had a significant deviation of neuropsychological functions from the normal population norms .
the functions of attention , verbal learning , and memory and visual working memory were commonly affected .
the present study noted that impairment in motor speed in 26.5% of patients , mental speed in 28.6% , focused attention in 36.7% , sustained attention in 58.3% , verbal fluency in 29.4% , response inhibition in 24.4% , visual working memory in 100% , and verbal working memory in 37.5% of patients . in verbal learning and memory , deficits were noted for verbal encoding in 56.2% , verbal retrieval in 56.2% , and verbal recognition in 72% .
three other patients had subtotal obliteration . based on the criteria defined by pollock and flickinger 12 patients ( 66.6% ) achieved an excellent outcome , i.e. obliteration of nidus and no new deficits [ figure 1a - d ] .
the outcome was fair in one patient ( obliteration and a major deficit ) . in three patients with subtotal
one other patient , who is a housewife who had transient hemiparesis , could resume light household activities .
the neuropsychological outcome was compared in those 18 patients who had evaluation both preprocedure and at the 2 years follow - up .
, patients showed an improvement in neuropsychological function at various categories . among them , the prominent statistically significant improvement was noted for set - shifting ability .
pretherapeutic median value for percentage perseverative responses was 26.5 and at follow - up , it reduced to 18.2 ( p = 0.039 ) .
set shifting improved in 11 patients ( 61.1% ) , remained same in 5 patients ( 27.7% ) , and deteriorated in two patients ( 11.1% ) .
other notable improvement was an increase in efficiency of planning in reduced time ( tested by the tower of london test ) .
patients took a median time of only 7.5 s as opposed to 16 s for successfully completing the 2 moves a section of the test ( p = 0.044 ) .
patients also showed improved ability for response inhibition at follow - up ( p = 0.003 ) .
the factors which are likely to influence the neuropsychological improvement namely volume of avm , location of the avm in the brain , spetzler - martin grade , radiosurgery based avm score , and marginal dose were evaluated for their impact on the outcome .
the effect of obliteration could not be analyzed because all avms showed some response at 2 years follow - up ( 15 patients showed total obliteration , and three had subtotal obliteration ) . similarly , the presence of intracranial hematoma also could not be analyzed as only two out of 18 patients had nonhemorrhagic presentation . on analysis ,
interestingly , patients with a higher spetzler - martin grade avm demonstrated a significantly more favorable shift in follow - up test values for set shifting function ( p = 0.021 ) .
post radiosurgery imaging changes have been defined as mild , moderate , and severe in previous studies .
five patients in the present series revealed imaging changes at various stages of follow - up [ figure 1e and f ] .
three patients had mild changes ; one had moderate pri , and one had severe pri . the mean duration for the appearance of pri after treatment was 10.8 5 months .
on assessing individual patient 's performance in relation to pri severity , it was noted that patients with postradiation imaging changes had a lesser tendency to improve in neuropsychological performance in follow - up tests .
four out of five patients with pri changes demonstrated some neuropyschological deterioration at follow - up evaluation .
one patient with moderate pri and two patients with mild pri showed deteriorated conceptual responses at follow - up .
one patient with severe pri showed deteriorated set shifting function on follow - up evaluation .
majority of patients with cerebral avms become symptomatic in the second through fourth decades of life with the most common presentation being intracranial hemorrhage ( 50% ) .
seizures are the second most common presentation which occur in 25% , followed by a headache or focal deficits in 15% .
the management options for patients diagnosed with intracranial avms include microsurgical resection , stereotactic radiosurgery , and embolization of avms .
embolization is frequently performed in conjunction with either surgical resection or radiosurgery but is rarely curative by itself .
the adverse effects of conventional radiotherapy on cognitive functioning , when administered for cancer treatment , are addressed in many studies .
a spectrum of late cognitive adverse effects can occur which include deficits in attention , working memory , learning ability , executive function in one end to incapacitating dementia in other end .
the effects are mediated by demyelination , microvascular injury , immunological response causing inflammatory reactions , atrophy of gray and white matter , altered neurotransmitter , and metabolites .
however , the effects on cognitive functions following conformal focused radiosurgical methods are not well documented . when radiosurgery is used to treat a benign condition like avm , taking into the fact that majority are in their young and active phase of life ,
concerns arise about possible radiation injury to the brain with its attendant late clinical and cognitive complications . of these complications ,
cognitive deficits may go underreported because they are not tested in detail in the routine clinical examination and are also rarely reported by patients .
this prompted us to do a prospective systematic study to look for the neuropsychological outcome of patients who underwent gkrs for brain avms .
it is known that patients with brain avms have a significant reduction in cognitive abilities compared with expected levels .
noted on pretherapeutic evaluation that 24% of patients had deficits in intelligence , 34% in attention and 48% in memory testing .
similarly , blonder et al . in a group of 10 patients noted below normal performance in most of the tests performed .
on analyzing the pretreatment data of 34 patients in the present series , baseline scores showed significant reduction compared to the normal population norms in majority of tests .
main deviations were noted in domains of focused attention ( by color trials in 36.7% ) , verbal memory ( by auditory verbal learning test in 3172% ) , verbal and visual working memory ( by n - back tests in 43.8% and 38.7% ) .
all the patients who underwent gkrs did not have any preprocedural complaints of cognitive dysfunction and were declared to be normal on routine clinical examination .
however , it was interesting to note that a majority of them had impaired cognitive dysfunction in one or more domains by formal neuropsychological assessment .
this fact underscores the incidence of subclinical cognitive dysfunction secondary to the disease in patients with avm , which often goes undetected . on follow - up , it was interesting to note that a statistically significant improvement was noted in certain domains of the tower of london test , wcst , stroop 's test .
no global deterioration was noted in any patient in any of these tests . by definition , planning is the identification and organization of the steps and elements needed to carry out an intention or achieve a goal .
the results in the present study indicate that patients have taken significantly less time following treatment with gkrs to plan simple tasks , as compared to their pretreatment status [ table 3 , the tower of london tests ] .
post - gkrs assessment indicated that the patients have significantly improved in terms of ability to form concepts as well as ability to shift sets where required [ table 3 , wcst ] .
the fact that 89% patients could return successfully to previous employment correlated well with the follow - up neuropsychological outcome .
the change in an occupation which was needed in two patients were due to motor deficits .
interestingly , a higher spetzler - martin grade was correlated with a favorable shift with improvement in follow - up test values .
the neuropsychological dysfunction in patients with avm before gkrs , mostly subclinical , can be attributed to the following factors : mass effect of avm , hematoma due to avm .
it can also be due to focal ischemia of the surrounding brain parenchyma due to steal phenomenon .
cognitive dysfunction can also be partly due to the use of anti epileptic drugs ( aeds ) in some patients as treatment of seizures . as evidenced in the present series , more than 50% of the patients with avm demonstrated cognitive dysfunction due to one or more of the above reasons .
most of the patients in this series had bled before presentation which can significantly contribute to the cognitive dysfunction .
it was interesting to note that a significant percentage improved in cognitive function , most probably due to the restoration of normal cerebral flow and reversal of ischemia to the surrounding brain .
higher the spetzler - martin grade or more complex the avm , it is more likely that cognitive deficits are due to avm per se and hence likely to show improvement at follow - up when they are obliterated .
we noted that the volume , location of the avm , radiosurgery based avm score , marginal dose , the presence of postradiation imaging ( pri ) changes were not found to influence the cognitive outcome .
postradiosurgery imaging changes ( pri ) is defined as development of new regions of t2 imaging changes on postradiosurgery follow - up mri scans irrespective of the presence of associated symptoms .
it is classified as mild when there is a perilesional rim of signal intensity changes , moderate when it is more than mild but less than one - fourth of the hemisphere is involved , severe when more than one - fourth of the hemisphere is involved .
however , the effect of these imaging changes in cognitive functions and correlation with the severity of neuropsychological deficits have not been evaluated previously . in the present study ,
five patients had pri changes ( three with mild changes , one had moderate pri , and one had severe pri ) . on observing individual patient 's performance , it was noted that patients with pri changes demonstrated deterioration in performance in follow - up tests . though no statistical significance could be demonstrated in view of very small numbers , it was interesting to note that 4/5 patients with pri revealed worsening of cognitive function , while most of the other patients indeed demonstrated improvement in cognition following gkrs , raising the possibility that the occurrence and severity of pri probably can affect cognitive outcome at follow - up .
overall , neuropsychological assessment indicates improvement on time taken to plan simple tasks , the formation of concepts , set shifting and response inhibition ( stroop test ) in the post- gkrs evaluation .
improvement may be due to normalization of perilesional perfusion disturbances and reduction in steal phenomenon with the obliteration of nidus .
overall enhanced neuronal plasticity along with the aforementioned changes might also have helped in the recovery of functions .
since the tests are repeated only after 2 years , a possibility of practice effect accounting for apparent improvement is nullified .
radiosurgery has no clinically harmful effect on cognitive and neuropsychological functioning in patients with brain avms . on the contrary
, there is an improvement in majority of the patients at 2 years following radiosurgery when the nidus is obliterated .
functions in which significant improvement noted are the time taken to plan tasks , ability to form concepts , as well as ability to shift sets where required and in response inhibition .
it is likely that that patients treated for avm with gkrs demonstrate improvement in neuropsychological functions unless they have radiation - induced changes effect of postradiation imaging changes on cognitive functions require further studies and such studies may have implications in calculating radiation dose for a volume , especially in repeat radiosurgery .
| background : radiosurgery is an alternative to surgical resection of arteriovenous malformation ( avm ) .
very few studies have addressed the concern of radiation injury to the brain and its attendant adverse effects on cognitive function.materials and methods : this prospective study included all patients who underwent gamma knife radiosurgery ( gkrs ) at our institute for cerebral avm between 2006 and december 2008 ( n = 34 ) .
all patients underwent neuropsychological evaluation before the procedure .
neuropsychological evaluation was repeated in eighteen patients 2 years following gkrs .
clinical outcome , avm obliteration , and factors influencing outcome were analyzed in these eighteen patients.results:before gkrs , more than 50% had significant impairment of neuropsychological functions compared to normal population norms .
66.6% achieved the excellent radiosurgical outcome . at
2 years follow - up , patients showed varied improvement in neuropsychological function in various categories .
pretherapeutic median value for percentage perseverative responses was 26.5 and at follow - up , it reduced to 18.2 ( p = 0.039 ) .
set shifting improved in 11 patients ( 61.1% ) , remained same in 5 patients ( 27.7% ) , and deteriorated in two patients ( 11.1% ) .
patients with a higher spetzler - martin grade avm demonstrated a significantly more favorable shift in follow - up test values for set shifting function ( p = 0.021 ) .
patients with postradiation imaging changes had lesser tendency to improve in neuropsychological performance at follow-up.conclusions:gkrs has no clinically harmful effect on cognitive and neuropsychological functioning in patients with brain avm . on the contrary
, there is an improvement in majority of patients at 2 years following radiosurgery when nidus is obliterated . |
torus palatinus ( tp ) and torus mandibularis ( tm ) are two of the most common intraoral exostoses .
other types of exostoses , affecting the palatal aspect of the maxilla ( palatal exostoses ) or the buccal aspects of the jaws ( buccal exostoses ) , are less commonly encountered .
tp is a sessile nodule of the bone found only in the midline of the hard palate , while tm is a bony protuberance located in the lingual aspect of the mandible , commonly at the canine and premolar areas .
although the tori are not pathologically significant , they may obscure radiographic details of maxillary sinuses and lower premolars .
they also interfere with the construction and function of removable dentures , as well as oral function and movement .
the prevalence of tori varies widely in different populations , ranging from 0.4% to 66.5% for tp and 0.5% to 63.4% for tm .
most authors reported that tp was more frequent in females , while tm affected males more than females .
the postulated causes include genetic factors that play a leading role in its occurrence , environmental factors , hyper
the present study was done to determine the prevalence , size , shape , and location of the tori and to investigate the sex and age relation of tp and tm in the malay population of malaysians .
our subjects consisted of 65 malays that included patients from dental outpatient clinic , students , staff , and dental auxiliaries of the school of dentistry , university sains malaysia , kubang kerian kelantan , malaysia .
the subjects were grouped into six different age groups : 1319 , 2029 , 3039 , 4049 , 5059 , and 60 years and above .
the presence of both tori was examined by clinical inspection and palpation performed by one examiner .
, study casts were made using alginate impression material for assessment of the size , shape , and location of the tori . for the size of tp ,
it was measured at the highest elevation of the outgrowth , using vernier calipers ( oem , china ) with the output to the nearest 0.01 mm .
the average size of the tori was graded according to the classification of reichart et al .
, as follows : small ( < 3 mm ) , medium ( 36 mm ) and large ( > 6 mm ) .
the location of tp was classified as premolar , molar , premolar to molar , incisor to premolar , and incisor to molar areas .
tm was identified by the number of nodes and their placement , and was divided into four categories : bilateral single , bilateral multiple , unilateral single , and unilateral multiple , as previously described by kolas et al .
location of tm was recorded as incisor , incisor to canine , incisor to premolar , incisor to molar , incisor and premolar , canine , and canine to premolar . the statistical package for social science ( version 11.0 ) was used for analysis .
the chi - square test was used to test for group differences and sex and age correlation with the prevalence of tp and tm .
tp and tm were observed in 50.8% and 4.6% respectively , for the total subjects [ tables 1 and 2 ] .
we are 95% sure that the prevalence of tp in population will be between 38% and 68% , and the prevalence of tm in population will be between 1% and 10% .
tp was found to be significantly higher in females than in males ( 90.9% versus 9.1% ; p < 0.05% ) , while tm was found to be not significantly different either in males or females ( 33.3% versus 66.7% ; p = 0.523 ) [ table 3 ] .
distribution of tp in relation to sexes distribution of tm in relation to sexes the distribution of tp and tm in relation to age and sexes out of 33 subjects with tp studied , we found that most were medium in size , followed by large in size , and the least was small in size ( 60.6% for medium in size ) .
the age and sex differences in the distribution pattern of tp according to size were statistically not significantly different ( p = 1.000 ) .
the age groups of subjects that mostly have medium size of tp were 2029 years old [ table 4 ] .
spindle shape of tp was the most frequently observed , followed by lobular , and the least was nodular .
there was a relationship between shape and size of tp ( p < 0.05 ) .
most spindle - shaped tp were in medium size , and most of the spindle - shaped tp were in 2029 years old age group .
the most common location for tp was in the premolar area and the least was in the incisor to molar area .
there was an association between age and location of tp ( p < 0.05 ) .
the distribution of tp in relation to sizes and age groups the distribution of tp in relation to shapes and age groups the distribution of tp in relation to location and age groups table 7 shows the distribution of tm in relation to the number of nodes and placement with age groups .
most tm were bilateral multiple ( 66.7% ) and followed by bilateral single ( 33.3% ) .
the most common location of tm was in the canine to premolar area ( 66.7% ) followed by incisor to premolar area ( 33.3% ) [ table 8 ] .
the distribution of tm in relation to shapes and age groups the distribution of tm in relation to location and age groups
tp and tm were observed in 50.8% and 4.6% respectively , for the total subjects [ tables 1 and 2 ] .
we are 95% sure that the prevalence of tp in population will be between 38% and 68% , and the prevalence of tm in population will be between 1% and 10% .
tp was found to be significantly higher in females than in males ( 90.9% versus 9.1% ; p < 0.05% ) , while tm was found to be not significantly different either in males or females ( 33.3% versus 66.7% ; p = 0.523 ) [ table 3 ] .
distribution of tp in relation to sexes distribution of tm in relation to sexes the distribution of tp and tm in relation to age and sexes
out of 33 subjects with tp studied , we found that most were medium in size , followed by large in size , and the least was small in size ( 60.6% for medium in size ) .
the age and sex differences in the distribution pattern of tp according to size were statistically not significantly different ( p = 1.000 ) .
the age groups of subjects that mostly have medium size of tp were 2029 years old [ table 4 ] .
spindle shape of tp was the most frequently observed , followed by lobular , and the least was nodular .
there was a relationship between shape and size of tp ( p < 0.05 ) .
most spindle - shaped tp were in medium size , and most of the spindle - shaped tp were in 2029 years old age group .
the most common location for tp was in the premolar area and the least was in the incisor to molar area .
there was an association between age and location of tp ( p < 0.05 ) .
the distribution of tp in relation to sizes and age groups the distribution of tp in relation to shapes and age groups the distribution of tp in relation to location and age groups
table 7 shows the distribution of tm in relation to the number of nodes and placement with age groups .
most tm were bilateral multiple ( 66.7% ) and followed by bilateral single ( 33.3% ) .
the most common location of tm was in the canine to premolar area ( 66.7% ) followed by incisor to premolar area ( 33.3% ) [ table 8 ] .
the distribution of tm in relation to shapes and age groups the distribution of tm in relation to location and age groups
the results of this study are in agreement with most of the previous studies that tp is more common in females than in males .
this is not in accordance with the results of this study . it can occur in both sexes , not particularly high in males only .
results from a previous study in thais suggest that the occurrence of tp is a sex - related phenomenon , but not for tm .
haugen stated that there was no obvious explanation for the gender differences but suggests genetics as a responsible factor . according to alvesalo et al . , who studied tm in females with turner syndrome ( 45 , x ) , it was suggested that sexual dimorphism in the manifestation of tm might result from the effect of the y chromosome on the growth , occurrence , expression , and timing of development of tm .
eggen and natvig reported a similar result in norwegians and summarized that decreased occurrence of tm among individuals over 50 years of age was related to the number of remaining teeth .
most of our subjects in the age group of 50 and above were partially edentulous .
so it may show that functional influences may be a contributing factor to the occurrence of the tori .
our result shows that the prevalence of tori was mostly in the second decade life , which is in accordance with the previous report of king and moore stated that , no great differences in the percentage of tor iwhich affected below the age of 30 and assumed that there was little or no further growth of tori after the age of 30 .
we found that subjects in the 2029 age group were more likely to have medium size of tp than those in the younger age group .
this result supports the association between age and continued growth of tori as reported by topazian and mullen .
10 study showed that females had higher frequency of tp and tended to have more medium in size tp than males , which is in accordance with the study in thais . in a singaporean study , chew and tan reported that 37% of tp present at two thirds of the palate and concluded that the chinese tended to have large tori .
most of the tp in our study was in the premolar to molar area like the previous study in thais .
some authors have reported that lobular tp was the rarest type . in the study in thais , flat tp was found to be the rarest type , and was in contrast with other studies in which this type of tp was predominant .
these studies are in accordance with most of the studies we also found that most tm were located at the canine to premolar area as in the previous reports . responding during life to environmental and functional factors that act in a complicated interplay with genetic factors , further studies are also needed to compare the prevalence of tori among races in malaysia .
the results of this pilot study need to be further studied to support the hypothesis that the occurrence of torus can be considered as a dynamic phenomenon , responding during life to environmental and functional factors that act in a complicated interplay with genetic factors .
further studies are also needed to compare the prevalence of tori among races in malaysia . | the aims of this study were to determine the prevalence , size , shape , and location of torus palatinus ( tp ) and torus mandibularis ( tm ) , and to assess their sex - related and age - related differences in the malay population .
sixty - five subjects were assessed for the presence of both tori at the school of dental sciences university sains malaysia .
the prevalence of tp was 3863% and that of tm was 110% .
tp was frequently more common in females than males ( 90.9% versus 9.1% ; p < 0.05 ) and was frequently found in medium sizes , spindle shaped , and was often located at the combined premolar to molar areas .
the prevalence of tm was not significantly different in males and females ( 33.3% versus 66.7% ; p = 0.523 ) , occurred most commonly in bilateral multiple form , and was often located at the canine to premolar area . |
epic - nl consists of the two dutch contributions to the epic study , the prospect - epic and morgen - epic cohorts .
these cohorts were set up simultaneously in 19931997 and merged into one dutch epic cohort . the design and rationale of epic - nl are described elsewhere ( 16 ) .
the prospect - epic study includes 17,357 women aged 4970 years living in utrecht and vicinity ( 17 ) .
the morgen - epic cohort consists of 22,654 adults aged 2164 years selected from random samples of the dutch population in three dutch towns ( 18 ) .
the study complies with the declaration of helsinki and was approved by the institutional board of the university medical center utrecht ( prospect ) and the medical ethical committee of tno nutrition and food research ( morgen ) .
after exclusion of those with prevalent diabetes ( n = 615 ) and of individuals with abnormal energy intake ( kcal < 600 or > 5,000 ) ( n = 108 ) , missing nutritional data ( n = 213 ) , and missing follow - up ( n = 981 ) , 38,094 participants were left for analysis .
daily nutritional intake was obtained from a self - administered food - frequency questionnaire ( ffq ) containing questions on the usual frequency of consumption of 79 main food items during the year preceding enrollment .
participants returned the ffq during the physical examination screening , where difficulties in filling out the questionnaire were discussed .
a registered dietitian checked the ffq for inconsistencies , which were resolved by contacting the participant .
the ffq has been validated against 12 24-h dietary recalls ( 19 ) with pearson correlation coefficients for protein intake of 0.67 in women and 0.71 in men ( 19 ) .
the glycemic index of foods , a measure of the extent to which foods raise the blood glucose level , was obtained from the foster - powell international table .
we calculated glycemic load by multiplying the glycemic index of a food with its carbohydrate content and then multiplied this value by the frequency of consumption of this food and summed the values over all food items .
intakes of nutrients were adjusted for total energy intake by the regression residual method and by using nutrient densities ( percentage of total energy intake , only for macronutrients ) ( 14 ) .
occurrence of diabetes during follow - up was self - reported in two follow - up questionnaires with 3- to 5-year intervals .
participants were asked whether diabetes was diagnosed , in what year , and by whom and what treatment was received . in the prospect study ,
incident cases of diabetes were detected via a urinary glucose strip test , sent out with the first follow - up questionnaire , for detection of glucosuria .
diagnoses of diabetes were also obtained from the dutch centre for health care information , which holds a standardized computerized register of hospital discharge diagnoses . in this
register , admission files have been entered continuously from all general and university hospitals in the netherlands from 1990 onward .
potential cases identified by any of these methods were verified against participants ' general practitioner or pharmacist information through mailed questionnaires .
diabetes was defined as being present when either of these confirmed the diagnosis . for 89% of participants with potential diabetes ,
verification information was available , and 72% were verified as having type 2 diabetes and were used for the analysis . at baseline ,
participants filled in a mailed general questionnaire containing questions on demographics , the presence of chronic diseases , and risk factors for chronic diseases .
smoking was categorized into current , past , and never smoker and parental history of diabetes into none , one , and both parents .
physical activity was assessed using a questionnaire validated in an elderly population ( 20 ) and categorized after calculating the cambridge physical activity score . because we could not calculate a total physical activity score for 14% of all participants
participants could return the questionnaire at the physical examination screening . during the baseline physical examination screening , systolic and diastolic blood pressure measurements
were performed twice in the supine position on the right arm using a boso oscillomat ( bosch & son , jungingen , germany ) ( prospect ) or on the left arm using a random zero sphygmomanometer ( morgen ) , from which the mean was taken .
hypertension was defined as being present when one or more of the following criteria were met : diastolic blood pressure 90 mmhg , systolic blood pressure 140 mmhg , self - reported antihypertensive medication use , or self - reported presence of hypertension .
weight during follow - up was derived from mailed follow - up questionnaires or physical examination ( doetinchem part ) .
weight change was defined as the difference between weight at baseline and follow - up . because the follow - up period varied , we calculated annual weight change by dividing weight change by the years of follow - up .
protein intake , adjusted for total energy intake by the regression residual method ( 14 ) , was categorized into quartiles .
cox proportional hazard models were used to calculate crude and adjusted hazard ratios ( hrs ) and 95% ci for the associations between quartiles of protein intake and diabetes .
we estimated ptrend by including median protein intakes per quartile as continuous variables in the cox regression models .
in addition , we analyzed associations between protein per 10 g of intake and diabetes risk . in the multivariate analysis , we first included sex ( male or female ) and age at recruitment ( continuous ) . in the second model , we added nutritional factors : energy - adjusted intake of saturated fat , monounsaturated fat , polyunsaturated fat , cholesterol , vitamin e , magnesium , fiber , and glycemic load ( continuous ) . in the third model
, we additionally corrected for diabetes risk factors : energy - adjusted alcohol consumption ( four categories ) , physical activity ( four categories ) , mean systolic and diastolic blood pressure ( continuous ) , education level ( three categories ) , and parental history of diabetes ( three categories ) . in the fourth model , bmi ( four categories ) and waist circumference ( continuous )
were included . to examine the influence of weight change during follow - up , we additionally corrected the analysis for annual weight change ( continuous ) .
we used a multivariate nutrient density model by including total energy intake and energy percentages of protein and other macronutrients in the multivariate cox regression model .
total energy intake was entered into the model to keep energy intake constant , essential for creating an isocaloric model ( 14 ) . by leaving out energy percentages from carbohydrate in the regression model
, we created a model in which the difference in diabetes risk associated with consumption of 5 energy % from protein at the expense of 5 energy % from carbohydrate , while total energy intake is kept constant , is presented .
similarly , by leaving out energy percentages from fat , we presented the difference in diabetes risk associated with consumption of 5 energy % from protein at the expense of 5 energy % from fat , while energy intake is held constant . interactions of protein intake with bmi ( < 25 or 25 kg / m ) and waist circumference ( < 84 or 84 cm ) were estimated using a likelihood ratio test and by including continuous interaction terms . the proportionality assumption was checked visually using log - minus - log plots , with no deviations detected .
daily nutritional intake was obtained from a self - administered food - frequency questionnaire ( ffq ) containing questions on the usual frequency of consumption of 79 main food items during the year preceding enrollment .
participants returned the ffq during the physical examination screening , where difficulties in filling out the questionnaire were discussed .
a registered dietitian checked the ffq for inconsistencies , which were resolved by contacting the participant .
the ffq has been validated against 12 24-h dietary recalls ( 19 ) with pearson correlation coefficients for protein intake of 0.67 in women and 0.71 in men ( 19 ) .
the glycemic index of foods , a measure of the extent to which foods raise the blood glucose level , was obtained from the foster - powell international table .
we calculated glycemic load by multiplying the glycemic index of a food with its carbohydrate content and then multiplied this value by the frequency of consumption of this food and summed the values over all food items .
intakes of nutrients were adjusted for total energy intake by the regression residual method and by using nutrient densities ( percentage of total energy intake , only for macronutrients ) ( 14 ) .
occurrence of diabetes during follow - up was self - reported in two follow - up questionnaires with 3- to 5-year intervals .
participants were asked whether diabetes was diagnosed , in what year , and by whom and what treatment was received . in the prospect study ,
incident cases of diabetes were detected via a urinary glucose strip test , sent out with the first follow - up questionnaire , for detection of glucosuria .
diagnoses of diabetes were also obtained from the dutch centre for health care information , which holds a standardized computerized register of hospital discharge diagnoses . in this
register , admission files have been entered continuously from all general and university hospitals in the netherlands from 1990 onward .
potential cases identified by any of these methods were verified against participants ' general practitioner or pharmacist information through mailed questionnaires .
diabetes was defined as being present when either of these confirmed the diagnosis . for 89% of participants with potential diabetes ,
verification information was available , and 72% were verified as having type 2 diabetes and were used for the analysis .
at baseline , participants filled in a mailed general questionnaire containing questions on demographics , the presence of chronic diseases , and risk factors for chronic diseases .
smoking was categorized into current , past , and never smoker and parental history of diabetes into none , one , and both parents .
physical activity was assessed using a questionnaire validated in an elderly population ( 20 ) and categorized after calculating the cambridge physical activity score . because we could not calculate a total physical activity score for 14% of all participants , we imputed missing scores using single linear regression modeling .
participants could return the questionnaire at the physical examination screening . during the baseline physical examination screening , systolic and
diastolic blood pressure measurements were performed twice in the supine position on the right arm using a boso oscillomat ( bosch & son , jungingen , germany ) ( prospect ) or on the left arm using a random zero sphygmomanometer ( morgen ) , from which the mean was taken .
hypertension was defined as being present when one or more of the following criteria were met : diastolic blood pressure 90 mmhg , systolic blood pressure 140 mmhg , self - reported antihypertensive medication use , or self - reported presence of hypertension .
weight during follow - up was derived from mailed follow - up questionnaires or physical examination ( doetinchem part ) .
weight change was defined as the difference between weight at baseline and follow - up . because the follow - up period varied , we calculated annual weight change by dividing weight change by the years of follow - up .
protein intake , adjusted for total energy intake by the regression residual method ( 14 ) , was categorized into quartiles .
cox proportional hazard models were used to calculate crude and adjusted hazard ratios ( hrs ) and 95% ci for the associations between quartiles of protein intake and diabetes .
we estimated ptrend by including median protein intakes per quartile as continuous variables in the cox regression models .
in addition , we analyzed associations between protein per 10 g of intake and diabetes risk . in the multivariate analysis ,
we first included sex ( male or female ) and age at recruitment ( continuous ) . in the second model , we added nutritional factors : energy - adjusted intake of saturated fat , monounsaturated fat , polyunsaturated fat , cholesterol , vitamin e , magnesium , fiber , and glycemic load ( continuous ) . in the third model
, we additionally corrected for diabetes risk factors : energy - adjusted alcohol consumption ( four categories ) , physical activity ( four categories ) , mean systolic and diastolic blood pressure ( continuous ) , education level ( three categories ) , and parental history of diabetes ( three categories ) . in the fourth model , bmi ( four categories ) and waist circumference ( continuous ) were included . to examine the influence of weight change during follow - up
we used a multivariate nutrient density model by including total energy intake and energy percentages of protein and other macronutrients in the multivariate cox regression model .
total energy intake was entered into the model to keep energy intake constant , essential for creating an isocaloric model ( 14 ) . by leaving out energy percentages from carbohydrate in the regression model
, we created a model in which the difference in diabetes risk associated with consumption of 5 energy % from protein at the expense of 5 energy % from carbohydrate , while total energy intake is kept constant , is presented .
similarly , by leaving out energy percentages from fat , we presented the difference in diabetes risk associated with consumption of 5 energy % from protein at the expense of 5 energy % from fat , while energy intake is held constant . interactions of protein intake with bmi ( < 25 or 25 kg / m ) and waist circumference ( < 84 or 84 cm ) were estimated using a likelihood ratio test and by including continuous interaction terms . the proportionality assumption was checked visually using log - minus - log plots , with no deviations detected .
mean protein intake was 75.7 g / day ; animal protein accounted for the majority .
the main contributors to protein intake were meat ( 39% ) , milk ( products ) ( 29% ) , and cheese ( 18% ) for animal protein and bread ( 43% ) , fruit and vegetables ( 14% ) , and potatoes ( 9% ) for vegetable protein .
moderate correlations were present between meat intake and total ( r = 0.30 ) and animal ( r = 0.36 ) protein , and between intake of milk ( products ) and total ( r = 0.46 ) and animal ( r = 0.50 ) protein . over the quartiles of total protein intake , mean age , bmi , waist circumference , and intakes of saturated fat and carbohydrates increased , whereas mean intakes of polyunsaturated fat and fiber and percentages of men , smokers , and physically inactive individuals decreased ( table 1 ) .
baseline characteristics of the study population , according to quartiles of daily nutritional total protein intake data are n ( % ) or means sd . * inactive according to cambridge physical activity index . during a mean follow - up of 10.1 1.9 ( mean sd ) years ,
adjustment for age , sex , dietary factors , and diabetes risk factors yielded an hr in the highest versus lowest quartile ( hrq4 ) of 1.67 ( 95% ci 1.292.16 ) . after further adjustment for adiposity measures ,
this association was no longer significant ( hrq4 1.18 [ 0.911.53 ] ) ( table 2 ) . removing either bmi or waist circumference from model 4 yielded comparable , nonsignificant associations ( excluding bmi , hrq4 1.22 [ 0.941.59 ] ) . for animal protein
10 g of intake showed comparable results , with significantly increased diabetes risk for higher total and animal protein intake in all models ( table 2 ) .
univariable and adjusted hrs ( 95% ci ) for the association between intake of protein , in quartiles and per 10 g , and incident type 2 diabetes data are hrs ( 95% ci ) . * significant at p < 0.001 level .
model 1 : corrected for sex ( male or female ) and age at recruitment ( continuous ) . model 2 : model 1 + energy - adjusted intake of saturated fat , monounsaturated fat , polyunsaturated fat , cholesterol , vitamin e , magnesium , fiber , and glycemic load ( continuous ) .
model 3 : model 2 + energy - adjusted alcohol consumption ( < 11 , 1125 , 2650 , or > 50 g / day ) , physical activity ( not active , moderately inactive , moderately active , or active ) , mean systolic and diastolic blood pressure ( continuous ) , education level ( high , middle , or low ) , and parental history of diabetes ( no , one parent , or both parents ) .
model 4 : model 3 + bmi ( < 20 , 2025 [ reference group ] , 2530 , or > 30
adjustment for weight change did not change these findings ( model 3 , hrq4 1.67 [ 1.282.16 ] ) . moreover , additional correction for meat and poultry intake in model 3 did not substantially change associations for either total or animal protein ( 1.50 [ 1.141.98 ] ) nor did adjustment for dairy intake ( 1.62 [ 1.242.11 ] ) .
excluding participants who followed a diet did not change the results ( model 3 , total protein 1.51 [ 1.112.06 ] ) nor did exclusion of participants with baseline cardiovascular disease , hypertension , or hyperlipidemia ( 1.68 [ 1.172.43 ] ) .
consumption of 5 energy % from protein at the expense of 5 energy % from fat increased diabetes risk , with an hr of 1.31 ( 95% ci 1.061.61 ) for each 5 energy % from protein exchanged for 5 energy % from fat in the final model . for consuming 5 energy % from protein at the expense of 5 energy % from carbohydrate
we observed no associations with consuming 5 energy % from vegetable protein ( table 3 ) .
multivariable hrs ( 95% ci ) for the association between the consumption of 5% energy from protein at the expense of 5% energy from fat or carbohydrates while keeping total energy intake constant and incident type 2 diabetes data are hrs ( 95% ci ) .
* model 3 : corrected for sex ( male or female ) , age at recruitment ( continuous ) , energy - adjusted intake of cholesterol , vitamin e , magnesium , fiber , and glycemic load ( continuous ) , total energy intake ( continuous ) , energy densities of fat , carbohydrates , and alcohol ( per 5 energy % ) , physical activity ( not active , moderately inactive , moderately active , or active ) , mean systolic and diastolic blood pressure ( continuous ) , education level ( high , middle , or low ) , and parental history of diabetes ( no , one parent , or both parents ) .
model 4 : model 3 + bmi ( < 20 , 2025 [ reference group ] , 2530 , or > 30 kg / m ) and waist circumference ( continuous ) .
we observed borderline significant interactions with bmi and waist circumference ( pinteraction = 0.08 for both ) in the relation between total protein and diabetes . for lean individuals , diabetes risk increased with increasing total protein intake ( hrq4 2.15 [ 95% ci 1.243.15 ] and 2.36 [ 1.304.29 ] for low bmi and waist circumference groups , respectively ) , whereas there was no relation in obese participants .
in addition , similar results were obtained when these interactions were analyzed continuously ( pinteraction < 0.05 ) .
correction for annual weight change did not change the associations ( low bmi group , total protein , hrq4 2.16 [ 1.253.75 ] ) .
in this study , high total and animal protein intake , but not vegetable protein intake , was associated with increased diabetes risk .
this relation was not explained by specific protein sources such as meat or by weight change during follow - up but was attenuated after adjustment for baseline adiposity measures .
consuming 5 energy % from protein at the expense of 5 energy % from carbohydrate or fat increased diabetes risk by 30% .
first , although we corrected for all possible available confounders , we can not exclude unknown or unmeasured confounding .
second , the presence of diabetes goes often undetected and may be preclinical up to 912 years ( 21 ) .
individuals with undetected diabetes may have been misclassified as nondiabetic individuals , resulting in attenuated associations .
strengths of our study include its prospective design , large sample size , and long follow - up .
use of validated cases of diabetes minimized the presence of false - positive cases of diabetes , reducing dilution of associations .
thus far , it is unclear whether a potential harmful effect of protein on diabetes is caused by high protein sources , such as meat , or by protein per se .
several studies related higher red , mainly processed , meat intake with increased diabetes risk ( 2,912 ) . when corrections for fat intake were made , associations remained ( 911 ) , indicating that the association is not caused by fat intake .
however , as most studies did not further address which nutrients were responsible for the increased diabetes risk with high meat intake , one can not conclude whether it is the protein or other nutrients in meat , such as iron , that promoted diabetes risk .
only one prospective study in women further investigated which nutrients in meat ( several types of fat and protein , heme , and total iron ) could promote diabetes ( 11 ) .
after correction for bmi , this association attenuated but remained significant , in contrast with our findings .
we observed that both high total and animal protein were associated with higher diabetes risk .
fat intake did not change much over the quartiles of protein intake , and the association was not altered after correction for fat intake . moreover , after correction for meat or dairy intake , the association between total and animal protein and diabetes remained , suggesting a detrimental role for protein per se in diabetes risk .
this association is further supported by the finding that consuming energy from protein at the expense of energy from either fat or carbohydrate increased diabetes risk .
we found no difference in risk when energy from protein was consumed at the expense of carbohydrate or fat , suggesting that the increase in protein itself and not the decrease in fat or carbohydrate caused this effect .
only one previous study , which focused on consuming carbohydrate at the expense of protein , reported similar findings ( 1 ) . yet , in that study , exchanging energy from protein for fat was not accounted for , and no differentiation was made for total protein content and protein source . because the majority of protein intake in our study is from animal sources , one might think the association with total protein is merely driven by the association with animal protein .
however , when we corrected the association between total protein and diabetes for animal protein , the association attenuated but remained ( model 3 , hrq4 1.46 [ 0.962.25 ] ) .
similarly , adjusting total protein for several sources of animal protein intake , such as meat , did not explain the entire association .
this finding indicates that part of the association between total protein and diabetes indeed seems to be explained by animal protein intake , but that a role for total protein can not be excluded . for vegetable protein
, we found an association in the same direction as that for animal protein , although this result did not reach statistical significance .
different effects of amino acids in animal and vegetable proteins on glucose metabolism may underlie the difference found between animal and vegetable protein ( 22,23 ) .
further studies addressing the effect of total protein intake in populations with differing intakes of protein sources are needed to establish the effects of total protein intake and specific protein sources on diabetes risk .
insulin resistance may arise , as amino acids can inhibit glucose transport and phosphorylation , leading to impaired glucose synthesis .
furthermore , amino acids intervene with glucose metabolism via stimulation of insulin and glucagon secretion and by serving as substrates for gluconeogenesis .
although stimulation of insulin secretion is expected to prevent hyperglycemia due to increased gluconeogenesis , this process might not sufficiently compensate in subjects with impaired insulin secretion ( 6,7 ) .
in contrast with our hypothesis , we only found an association in lean individuals . in the epic - potsdam study , a similar but nonsignificant interaction with adiposity
a recent study showed that soluble transferrin receptor was inversely associated with insulin sensitivity only in normal glucose tolerant and lean individuals , suggesting a mechanism through iron metabolism ( 24 ) .
iron overload is associated with increased diabetes risk ( 25 ) . because increased animal protein intake may contribute to increased body iron load , the association between high ( animal ) protein intake and diabetes in the nonobese people
however , markers of body iron load ( serum ferritin , iron , total iron binding capacity , and transferrin saturation ) could not explain this association in a random sample of our cohort ( data not shown ) .
second , it is unlikely that weight gain during follow - up explains the increased diabetes risk in nonobese individuals , as correction for annual weight change did not change these findings .
third , the associations between protein and diabetes were largely attenuated after correction for adiposity measures , raising the possibility for adiposity to be an intermediate in this relationship . however , when we adjusted the association between protein intake and diabetes for bmi ( continuous ) , the positive association in the lean group remained present , indicating that this possibility is unlikely . finally ,
because of the direction of the interactions , borderline significance , and relatively few nonobese diabetic participants , we can not exclude the possibility that these interactions are due to chance . in summary ,
our findings also suggest a similar association for protein itself instead of only animal sources . the consumption of energy from protein , at the expense of the same percentage of energy from either fat or carbohydrate , increased diabetes risk by 30% . more research into the effect of total protein intake in different populations with intakes of protein from different sources is needed to establish the effects of total protein intake and differing sources on diabetes risk . yet , these results underline the importance of taking into account the protein content of diet in dietary recommendations to prevent diabetes . | objectivedietary recommendations are focused mainly on relative dietary fat and carbohydrate content in relation to diabetes risk . meanwhile
, high - protein diets may contribute to disturbance of glucose metabolism , but evidence from prospective studies is scarce .
we examined the association among dietary total , vegetable , and animal protein intake and diabetes incidence and whether consuming 5 energy % from protein at the expense of 5 energy % from either carbohydrates or fat was associated with diabetes risk.research design and methodsa prospective cohort study was conducted among 38,094 participants of the european prospective investigation into cancer and nutrition ( epic)-nl study .
dietary protein intake was measured with a validated food frequency questionnaire .
incident diabetes was verified against medical records.resultsduring 10 years of follow - up , 918 incident cases of diabetes were documented .
diabetes risk increased with higher total protein ( hazard ratio 2.15 [ 95% ci 1.772.60 ] highest vs. lowest quartile ) and animal protein ( 2.18 [ 1.802.63 ] ) intake .
adjustment for confounders did not materially change these results . further adjustment for adiposity measures attenuated the associations .
vegetable protein was not related to diabetes .
consuming 5 energy % from total or animal protein at the expense of 5 energy % from carbohydrates or fat increased diabetes risk.conclusionsdiets high in animal protein are associated with an increased diabetes risk .
our findings also suggest a similar association for total protein itself instead of only animal sources .
consumption of energy from protein at the expense of energy from either carbohydrates or fat may similarly increase diabetes risk .
this finding indicates that accounting for protein content in dietary recommendations for diabetes prevention may be useful . |
given the types of intermittent maximum effort needed during a team sport such as handball , it is important to evaluate performance by measuring variables such as muscle strength , velocity and resistance ( marques , 2010 ; michalsik et al . , 2013 ; pvoas et al . ,
the factors that determine performance in handball are explosive actions such as jumping ( i.e. , vertical or horizontal ) , speed of reduced - space displacements ( i.e. , 10 and 20 m sprint time ) and ball release velocity ( brv ) .
to assess physical performance of a handball player , several tests are commonly used encompassing sprinting , jumping , agility and maximal strength as often measured in the one repetition maximum ( 1rm ) , or ball release velocity tests ( marques and gonzlez badillo , 2006 ) .
thus , according to the scores obtained in these tests , athletes can be classified in terms of a particular skill ( e.g. , the player who is able to throw the ball faster or lift the heaviest weight in the bench press ) ( ingebrigtsen et al . , 2013 ; vila et al . , 2012 )
the problem with this type of classification is that , despite an existing correlation between the maximum weight lifted in the bench press and ball throwing velocity ( gorostiaga et al .
, 2005 ; granados et al . , 2007 ; marques et al . , 2007 ) , the player who throws the ball more rapidly will not necessarily be the individual attaining the highest 1rm in the bench press .
the second objective of such tests is to compare the effects of training programs on performance during a given period of time ( gorostiaga et al . , 1999 ) or during a competition season ( bresciani et al . , 2010 ;
; marques et al . , 2007 ) , the player who throws the ball more rapidly will not necessarily be the individual attaining the highest 1rm in the bench press . the second objective of such tests is to compare the effects of training programs on performance during a given period of time ( gorostiaga et al . , 1999 ) or during a competition season ( bresciani et al . , 2010 ;
a search of the scientific literature indicates that such measures of performance are commonly used to determine the response to a given training intervention ( chelly et al . , 2009 , 2013 ;
the researcher looks for differences between pre- and post test values of a certain variable .
moreover , test scores may be used to measure the effect size produced by a training intervention on a study population ( peterson et al . , 2004 ; rhea et al . , 2003
when used in this manner , the cross - sectional perspective of these tests is completely lost . in other words ,
this information could be of vital importance to measure the physical state of a player before a training program compared with the rest of the players .
this line of research has generated many studies in different sports such as soccer ( unnithan et al .
, 2012 ; votteler and hner , 2013 ; william and reilly , 2000 ) , handball ( baker et al . , 2013 ; lidor et al . , 2005 ; mohamed et al . , 2009 ) , basketball ( hoare , 2000 ) or rugby ( gabbett , 2006 , 2011 ) in which the principle objective was to differentiate elite from amateur players based on variables related to physical performance , anthropometric measures or decision making ability
( 2011 ) developed a multidisciplinary model to identify talented young handball players ( under-14 [ u14 ] , under-16 [ u16 ] and under-18 [ u18 ] ) .
results indicated that elite players could be distinguished from non - elite ones according to physical performance characteristics , although the discrimination capacity of this method varied according to the age group tested .
( 2011 ) clustering of handball players occurred naturally depending upon their respective team ( elite vs. non - elite team ) and age ( u14 , u16 and u18 ) . using multivariate statistics ( i.e. , cluster analysis ) and theoretical knowledge about performance , it is possible to create an objective physical performance classification system of the handball players .
therefore , the aim of the present study was to identify different cluster groups of handball players according to their physical performance level evaluated in a series of physical assessments ( a countermovement jump [ cmj ] , a squat jump [ sj ] , 10 m sprint time [ st10 ] , 20 m sprint time [ st20 ] , the one repetition maximum [ 1rmest ] , peak power at the propulsive phase [ ppeakmp ] and mean power at the propulsive phase [ pmppmp ] in the bench press and ball release velocity [ brv ] ) , which could then be used to design a training program based on individual strengths and weaknesses , and to determine which of these variables best identified elite performance in a group of under-19 [ u19 ] national level handball players .
participants age , body mass and height ( mean sd ) were 18 0.4 years , 87.38 9.97 kg and 189 6.72 cm , respectively .
written informed consent was obtained from the participants after the nature of the study and its objectives had been explained .
the study protocol received university of granada review board approval and was performed in accordance with the tenets of the declaration of helsinki .
a cross - sectional design was performed to measure physical performance variables in young handball players .
this investigation was conducted one month prior to the european junior handball championship in 2013 . using a multivariate approach ,
a hierarchical cluster analysis was performed to create a grouping of training based on different important training variables in handball players .
after that , a descriptive discriminant analysis was performed to indicate the most important variable to generate this grouping classification .
the measured variables included in the analysis were in vertical dimension ( cmj and sj ) , in horizontal dimension ( st10 and st20 ) and cross - sectional dimension ( 1rmest , ppmppmp and pmppmp in the bench press and brv ) .
the physical tests were performed during the training camp for the european junior handball championship in 2013 .
sprint time , brv , cmj , sj and incremental - load bench press tests were applied since these are regularly used in testing physical performance in handball and measure variables such as muscle strength , power and velocity in the upper and lower body very well ( granados et al .
, 2008 ; ingebrigtsen et al . , 2013 ; krger et al . , 2014 ; marques and gonzlez badillo , 2006 ) .
these physical performance tests were conducted at the team s playing court and training facilities . before the tests , participants attended a familiarization session in which each test protocol and its key points were explained in detail .
all participants undertook their standard 15 min warm up protocol involving sprints with direction changes and ball passes .
after a 5 min rest period in which the subjects could drink water ad libitum , they completed the physical performance assessment tests established for the session .
the tests were performed over 3 days as follows : day 1 , st10 and st20 and brv ; day 2 , cmj and sj ; and day 3 , an incremental - load bench press test .
at least 24 h elapsed between each testing session .
sprint tests : a photocell system ( kit racetime 2 light radio , microgate , italy ) was used to measure the time ( s ) needed to cover 10 m and 20 m distances .
the starting point was 1 m before the position of the photocell so that each subject selected the moment of starting .
each subject was allowed three attempts at the test and players received feedback of the score obtained in every sprint .
descriptive variables recorded ( provided as their mean , standard deviation , intraclass correlation coefficient [ icc2.k ] , mean 95% ci icc2.k and repeated measures analysis of variance [ rm anova ] ) .
value used was the mean of repetitions 2 and 3 ( see procedures for details ) ppeakmp = peak power in the maximum power load .
= intraclass correlation coefficient ( icc2.k ) = only one measure of this variable used .
vertical jump tests : the vertical jump height tests ( i.e. , cmj and sj ) were performed using a jumping mat ( optojump , microgate , italy ) , which measures the time the feet are off the mat and translates the result into a jump height in cm .
the technique was explained before each test and each athlete had 3 attempts at each jump .
the sj was started from a static semisquatting position with the knee angle of 90 degrees of flexion , followed by subsequent action , during which the leg and hip extensor muscles contracted concentrically . at the beginning of the cmj test , each participant stood erect on the jump mat .
the test consisted of preparatory movement down to approximately 90 degrees of knee flexion , stretching the leg extensor muscles followed by explosive maximal extension in the opposite direction .
for the cmj , the mean height of the last 2 attempts was included in the analysis while for the sj , the mean of the last 3 attempts ( i.e. , repetitions 2 and 3 ) were used ( table 1 ) .
ball release velocity test : brv was determined with a three - pace run up .
peak velocity was measured using a radar stalker ats gun ( radar sales , minneapolis , mmn , usa ) positioned behind the goal . just after each throw
the mean of repetitions 2 and 3 was entered in the analysis ( table 1 ) .
upper body force - velocity test : in the last testing session , athletes performed an incremental - load free bench press test ( salter ) .
the bench position , barbell grip and displacement were previously established and controlled in each attempt or repetition .
participants performed a 15 min warm up and 2 sets of presses with a light load ( i.e. , barbell weight of 20 kg ) before starting the test protocol .
the starting weight for the test was 25 kg and was increased by 10 kg up to a load of 85 kg .
three repetitions were performed at each intensity . during the incremental test , the descent phase of the bar was controlled by verbal instructions from the researcher as follows : down ( 2 s ) , chest
( 1 s ) , go . to avoid the rebound effect , the
each subject was encouraged to lift the barbell as quickly as possible during the concentric phase ( i.e. , explosively ) until the elbows were completely extended .
a linear displacement system was employed ( t - force system , ergotech , murcia , spain ) to record the mean velocity of the propulsive phase , mean power of the propulsive phase and the estimated 1 repetition maximum ( 1rmest ) .
it had been shown that mean velocity could be used to precisely estimate loading intensity ( gonzlez - badillo and snchez - medina , 2010 ) .
information of the movement velocity of the bar is transmitted and transformed from analogue to digital by a data acquisition board .
vertical instantaneous velocity was directly sampled by the device at that the frequency of 1000 hz .
the propulsive phase was defined as the portion of the concentric phase during which the measured acceleration ( a ) was greater than acceleration due to gravity ( i.e. , a > -9.8 ms ) ( gonzlez - badillo and snchez - medina , 2010 ) . once the test was completed and the data processed , the load corresponding to the mean maximum power ( loadmp ) was determined for each individual .
then , for this load , we calculated the variables of mean power of the propulsive phase ( pmppmp ) and the peak power ( ppeakmp ) .
the variables were transformed into the z - score in order to analyse the presence of outliers .
the intrasession intraclass correlation coefficient was calculated for all tests ( except for the 1rmest variable ) using the equation 2.k ( icc2.k ) ( weir , 2005 ) .
the coefficient of variation ( cv ) was calculated as 100(e 1 ) ( hopkins , 2011 ) .
the strength of a correlation was defined as r = 0 0.1 ( trivial ) , r = 0.1 0.3 ( small ) , r = 0.3 0.5 ( moderate ) , r = 0.5 0.7 ( large ) , r = 0.7 0.9 ( very large ) and r = 0.9 1 ( nearly complete ) ( hopkins , 2003 ) .
a repeated measures analysis of variance ( rm anova ) was used to assess differences in the means recorded for the three repetitions of each dependent variable .
when the sphericity test was violated , a greenhouse - geisser estimation was used to adjust the degree of freedom .
we used hierarchical cluster analysis to create several groupings according to the results of the performance tests .
each variable was transformed into the z - score to avoid effects of different measurement scales . as a measure of similarity ,
the clustering ward s method was used . a two between group multivariate analysis of variance ( manova )
was used for upper trunk ( 1rmest , ppeakmp , pmppmp and brv ) and lower trunk ( st10 , st20 , cmj and sj ) variables , where the predictor variable was the cluster solution ( i.e. , cluster 1 , 2 , 3 and 4 ) .
the players were classified into different clusters through a descriptive discriminant analysis . as the criterion variable
, we used the 4-cluster classification generated by the cluster analysis , while lower trunk ( st10 , st20 , cmj and sj ) and upper trunk ( 1rmest , ppeakmp , pmppmp and brv ) variables were entered as the predictor variables .
all statistical analyses were performed using spss software ( spss inc , illinois , usa ) .
participants age , body mass and height ( mean sd ) were 18 0.4 years , 87.38 9.97 kg and 189 6.72 cm , respectively .
written informed consent was obtained from the participants after the nature of the study and its objectives had been explained .
the study protocol received university of granada review board approval and was performed in accordance with the tenets of the declaration of helsinki .
a cross - sectional design was performed to measure physical performance variables in young handball players .
this investigation was conducted one month prior to the european junior handball championship in 2013 . using a multivariate approach ,
a hierarchical cluster analysis was performed to create a grouping of training based on different important training variables in handball players .
after that , a descriptive discriminant analysis was performed to indicate the most important variable to generate this grouping classification .
the measured variables included in the analysis were in vertical dimension ( cmj and sj ) , in horizontal dimension ( st10 and st20 ) and cross - sectional dimension ( 1rmest , ppmppmp and pmppmp in the bench press and brv ) .
the physical tests were performed during the training camp for the european junior handball championship in 2013 .
sprint time , brv , cmj , sj and incremental - load bench press tests were applied since these are regularly used in testing physical performance in handball and measure variables such as muscle strength , power and velocity in the upper and lower body very well ( granados et al . , 2008 ;
these physical performance tests were conducted at the team s playing court and training facilities . before the tests
, participants attended a familiarization session in which each test protocol and its key points were explained in detail .
all participants undertook their standard 15 min warm up protocol involving sprints with direction changes and ball passes .
after a 5 min rest period in which the subjects could drink water ad libitum , they completed the physical performance assessment tests established for the session .
the tests were performed over 3 days as follows : day 1 , st10 and st20 and brv ; day 2 , cmj and sj ; and day 3 , an incremental - load bench press test .
sprint tests : a photocell system ( kit racetime 2 light radio , microgate , italy ) was used to measure the time ( s ) needed to cover 10 m and 20 m distances .
the starting point was 1 m before the position of the photocell so that each subject selected the moment of starting .
each subject was allowed three attempts at the test and players received feedback of the score obtained in every sprint .
descriptive variables recorded ( provided as their mean , standard deviation , intraclass correlation coefficient [ icc2.k ] , mean 95% ci icc2.k and repeated measures analysis of variance [ rm anova ] ) .
value used was the mean of repetitions 2 and 3 ( see procedures for details ) ppeakmp = peak power in the maximum power load .
= intraclass correlation coefficient ( icc2.k ) = only one measure of this variable used .
vertical jump tests : the vertical jump height tests ( i.e. , cmj and sj ) were performed using a jumping mat ( optojump , microgate , italy ) , which measures the time the feet are off the mat and translates the result into a jump height in cm .
the technique was explained before each test and each athlete had 3 attempts at each jump .
the sj was started from a static semisquatting position with the knee angle of 90 degrees of flexion , followed by subsequent action , during which the leg and hip extensor muscles contracted concentrically . at the beginning of the cmj test , each participant stood erect on the jump mat .
the test consisted of preparatory movement down to approximately 90 degrees of knee flexion , stretching the leg extensor muscles followed by explosive maximal extension in the opposite direction .
for the cmj , the mean height of the last 2 attempts was included in the analysis while for the sj , the mean of the last 3 attempts ( i.e. , repetitions 2 and 3 ) were used ( table 1 ) .
ball release velocity test : brv was determined with a three - pace run up .
peak velocity was measured using a radar stalker ats gun ( radar sales , minneapolis , mmn , usa ) positioned behind the goal . just after each throw
the mean of repetitions 2 and 3 was entered in the analysis ( table 1 ) .
upper body force - velocity test : in the last testing session , athletes performed an incremental - load free bench press test ( salter ) .
the bench position , barbell grip and displacement were previously established and controlled in each attempt or repetition .
participants performed a 15 min warm up and 2 sets of presses with a light load ( i.e. , barbell weight of 20 kg ) before starting the test protocol .
the starting weight for the test was 25 kg and was increased by 10 kg up to a load of 85 kg .
three repetitions were performed at each intensity . during the incremental test , the descent phase of the bar was controlled by verbal instructions from the researcher as follows : down ( 2 s ) , chest ( 1 s ) , go . to avoid the rebound effect , the
each subject was encouraged to lift the barbell as quickly as possible during the concentric phase ( i.e. , explosively ) until the elbows were completely extended .
a linear displacement system was employed ( t - force system , ergotech , murcia , spain ) to record the mean velocity of the propulsive phase , mean power of the propulsive phase and the estimated 1 repetition maximum ( 1rmest ) .
it had been shown that mean velocity could be used to precisely estimate loading intensity ( gonzlez - badillo and snchez - medina , 2010 ) .
information of the movement velocity of the bar is transmitted and transformed from analogue to digital by a data acquisition board .
vertical instantaneous velocity was directly sampled by the device at that the frequency of 1000 hz .
the propulsive phase was defined as the portion of the concentric phase during which the measured acceleration ( a ) was greater than acceleration due to gravity ( i.e. , a > -9.8 ms ) ( gonzlez - badillo and snchez - medina , 2010 ) .
once the test was completed and the data processed , the load corresponding to the mean maximum power ( loadmp ) was determined for each individual .
then , for this load , we calculated the variables of mean power of the propulsive phase ( pmppmp ) and the peak power ( ppeakmp ) .
the variables were transformed into the z - score in order to analyse the presence of outliers .
the intrasession intraclass correlation coefficient was calculated for all tests ( except for the 1rmest variable ) using the equation 2.k ( icc2.k ) ( weir , 2005 ) .
the coefficient of variation ( cv ) was calculated as 100(e 1 ) ( hopkins , 2011 ) .
pearson correlation coefficients were used to determine interrelationships between variables . the strength of a correlation was defined as r = 0 0.1 ( trivial ) , r = 0.1 0.3 ( small ) , r = 0.3 0.5 ( moderate ) , r = 0.5 0.7 ( large ) , r = 0.7 0.9 ( very large ) and r = 0.9 1 ( nearly complete ) ( hopkins , 2003 ) .
a repeated measures analysis of variance ( rm anova ) was used to assess differences in the means recorded for the three repetitions of each dependent variable .
when the sphericity test was violated , a greenhouse - geisser estimation was used to adjust the degree of freedom .
we used hierarchical cluster analysis to create several groupings according to the results of the performance tests .
each variable was transformed into the z - score to avoid effects of different measurement scales . as a measure of similarity ,
a two between group multivariate analysis of variance ( manova ) was used for upper trunk ( 1rmest , ppeakmp , pmppmp and brv ) and lower trunk ( st10 , st20 , cmj and sj ) variables , where the predictor variable was the cluster solution ( i.e. , cluster 1 , 2 , 3 and 4 ) . the players were classified into different clusters through a descriptive discriminant analysis . as the criterion variable , we used the 4-cluster classification generated by the cluster analysis , while lower trunk ( st10 , st20 , cmj and sj ) and upper trunk ( 1rmest , ppeakmp , pmppmp and brv ) variables were entered as the predictor variables .
all statistical analyses were performed using spss software ( spss inc , illinois , usa ) .
table 1 provides the means ( sd ) , the 95% confident interval ( ci ) of the mean , icc 2.k and 95% ci icc2.k of the variables recorded ( lower trunk [ st10 , st20 , cmj and sj ] and upper trunk [ 1rmest , ppeakmp , pmppmp and brv ] ) . in parallel , the 3 repetitions of each test and the variables examined were compared by rm anova .
significant differences were detected for the cmj ( f [ 1.43 , 21.41 ] = 2.2 , p = .021 , p = .13 ) and brv ( f [ 2 , 30 ] = 8 , p = .004 , p = .35 ) ; post hoc bonferroni correction indicated differences between set 1 with 2 ( p = .01 ) for the cmj variable and set 1 vs set 2 ( p = .020 ) and set 1 vs. set 3 ( p = .023 ) in brv .
thus , we used the mean of set 2 and 3 for the final values of brv and the cmj . for the rest of the variables the mean of the three attempts
pearson correlation coefficients between brv and bench press variables ( 1rmest , ppeakmp and pmppmp ) were respectively .68 ( p < .01 ) large , .55 ( p < .05 ) large and 0.54 ( p < .05 ) large .
the hierarchical cluster analysis dendrogram constructed before examining any groupings is provided in figure 1 . in the analysis , each subject was first considered as an individual group i.e. , 16 subjects = 16 groups , in which each of the nine variables was analysed .
then , differences between all subjects were analysed with each subject assigned to the same group in a cluster matrix .
dendrogram generated by the cluster analysis . after creating a grouping classification using a cluster analysis , the players were classified in 4 clusters .
the means sds of all the variables of the grouped solution generated by the cluster analysis are provided in table 2 .
mean sd of the performance indicators according to the clusters generated by the hierarchical cluster analysis .
first multivariate statistical technique used ( i.e. , cluster analysis ) provided a grouping classification of the players based on a measure of similarity ( i.e. , squared euclidean distance ) and the agglomeration method ( i.e. , wards method ) .
, a manova showed significant differences in the lower ( = .07 ; p = .004 ; p = .59 ) and upper trunk ( = .03 ; p = .0001 ; p = .69 ) .
figure 2 shows z - score values of each variable ( i.e. , the upper and lower trunk ) at each cluster solution obtained .
z - scores of each variable at both the upper ( a ) and lower ( b ) trunk at each cluster .
the following values were obtained from discriminant analysis : function 1 ( = .001 ; p = .0001 ) , function 2 ( = .044 ; p = .013 ) and function 3 ( = .24 ; p = .046 ) .
the percentage of variance explained by each of these functions was 75% , 15% and 10% , respectively .
combined intra - group correlations between the discriminant variables and the typified canonical discriminant functions were r = .44 and -.39 ( brv and st20 for function 1 , respectively ) , r = .50 ( 1rmest for function 2 ) and r = .48 , r = .47 , r = -.42 , -.36 and .22 ( pmpfmp , ppeakmp , cmj , sj and st10 for function 3 , respectively ) .
these variables at each canonical function reflected the most important variable to classify players in the cluster solutions .
discriminant analysis provided a probability of each player and their percentage probabilities of belonging to each cluster .
this analysis revealed that 100% of cases were correctly classified in the 4-group solution generated by the cluster analysis using the performance variables assessed .
figure 3 shows the mean of the centroids ( square ) and the groupings of different players in their corresponding cluster ( circle ) .
graphical representation of each player ( circle ) and the centroid mean for each group ( square ) .
a qualitative analysis showed that players were distributed according to their playing positions as follows : cluster 1 ( two center and two right back players ) ; cluster 2 ( one center , one left wing , two left backs , one right back and one pivot ) ; cluster 3 ( one goalkeeper , one right back and one left wing ) and cluster 4 ( two pivots and one goalkeeper ) .
the aim of the present study was to identify different cluster groups of handball players according to their physical performance level assessed in a series of tests , which could then be used to design a training program based on the players individual needs .
moreover , our study sought to determine the variables that best reflected elite performance of a group of male handball players in the ui9 national team .
to the best of our knowledge , this is the first attempt to create a classification scheme that analyses individual strengths and weaknesses in elite handball players based on their physical performance using a multivariate statistical approach .
several studies have compared the performance of individual athletes considering a given skill or variable ( e.g. , brv , 1rm in the bench press , rfd , repeated sprint ability ) ( krger et al . , 2014 ; marques et al . , 2011 ; michalsik et al . ,
physiologic variables interact and can obscure which factors are the real determinants of performance . in handball , factors such as brv ( marques et al . , 2007 ) , training with external resistance ( hermassi et al . ,
2011 ) or combining different training modalities ( van den tillaar , 2004 ) seem to play an important role in the physical performance of players . in the present study , physical performance
was assessed at two levels : the upper trunk ( 1rmest , ppeakmp , pmppmp and brv ) and the lower trunk ( cmj , sj , st10 and st20 ) . before classifying players according to their physical performance , a cluster analysis was performed to identify individuals obtaining similar scores in the performance tests used , i.e. , according to their strengths and weaknesses . as it may be seen in the dendrogram generated by the cluster analysis ( figure 1 ) , we opted for a 4-cluster solution .
table 2 provides the means and sds of each performance variable analysed according to the cluster each player was assigned to .
this information could be useful in order to create different training groups based on physical weakness points .
in other words , using this statistical approach makes it possible to provide specific training to each group of players and enhance the physical performance of the players based on their specific needs .
this allows for the identification of strengths and weaknesses in the three physical assessment dimensions ( e.g. , cluster 3 should focus on improving cross - sectional and horizontal dimension performance and cluster 2 should improve vertical dimension performance ) ( figure 2 ) .
therefore , once the identification of strengths and weaknesses was performed , it was possible to design specific training sessions for each cluster solution .
it should be mentioned that the cluster analysis allocates the same weight to each performance variable examined .
thus , once all the players were grouped according to their physical performance , a discriminant analysis was used to determine whether the players could be classified in the established groups according to the performance variables tested and to check the specific weight of each variable for this purpose .
this classification is graphically shown in figure 3 , which represents the organization of the 16 players assessed . in figure 3
it may be observed how the cluster analysis generated four extremely homogeneous groups in themselves , but these were heterogeneous compared to each other ( each square represents the mean of the centroids of each group ) . in addition , discriminant analysis generated three discriminant canonical functions .
analysis of these functions demonstrated statistical significance in all cases when classifying clusters of players .
moreover , the first and second functions have greater discriminative power between players classification ( 75% and 15% , respectively ) . as it may be seen in the results section , in the first canonical function ( which was the function that explained more variance ) , brv was the variable with most discriminatory power for the upper trunk , and st20 for the lower trunk .
our results support these of previous literature suggesting that brv is often considered as a key performance variable in handball players ( chelly et al .
, 2010 ; hermassi et al . , 2011 ; marques and gonzlez badillo , 2006 ) . in the second canonical discriminating function ,
this variable has a lot of significance as several studies have shown the importance of maximum dynamic strength to improve 1rmest in handball players .
a large correlation coefficient was obtained between brv and 1rmest ( r = .68 , p < .01 ) .
as described before , it seems that these variables usually can be described by a positive linear function ( chelly et al .
, 2010 ; marques , 2006 ; marques and gonzlez - badillo , 2006 ) .
finally , in the last canonical discriminatory function , the best performance indicators to correctly classify the players were ppeakmp , pmppmp , cmj , sj and st10 .
as mentioned earlier , no prior study had examined ppeakmp and pmppmp variable in handball players , although marques et al .
( 2007 ) correlated the peak power attained during the bench press ( 26 and 36 kg ) with brv ( r = .63 , p = .017 ) .
however , in our study these variables ( pmppmp and ppeakmp ) returned a large coefficient with brv ( r = .55 , p < .05 and r = .54 , p < .05 , respectively ) .
the main limitation of our study was the sample size ( n = 16 ) , although due to the selective nature of the elite population used , this was the maximum sample size available .
however , our intention was not to generalize the results to the population , but to create a multivariate systematic tool to assess intra - squad physical performance .
it can be therefore applied in order to acknowledge which variables require a training focus within the squad for certain individuals .
it is interesting to note that this is the first study that uses a multivariate approach to analyse physical performance with a practical application .
information obtained in other age groups will help generate a broader database to classify potential players .
this will help identify new performance measures that will be able to better discriminate between players whose physical performance is optimal in a given dimension .
also future research needs to be conducted in order to make a cluster and discriminant analysis in the specific handball playing positions and moreover , including other types of variables ( i.e. , decision - making and endurance tests ) . this would allow identification of the most relevant strength training variables at each specific playing position .
the present study provides new information on specific interactions between physical performance and cluster created solutions . according to performance measured in upper body variables ( 1rmest , ppeakmp , pmppmp and brv ) and others such as 10 m and 20 m sprint velocity , as well as cmj and sj height
, we were able to classify 16 young male elite handball players into four groups through cluster analysis .
the results revealed that within a young elite handball team there were various physical performance levels and therefore , these athletes required different training focuses based on their individual strengths and weaknesses .
finally , a discriminant analysis identified brv , st20 , 1rmest , ppeakmp and pmppmp as the variables that best reflected elite physical performance in the handball players .
the information emerging from this study can help researchers , coaches and strength and conditioning coaches objectively evaluate the results of physical fitness tests conducted on the players in their teams .
notably , the classification system obtained through cluster and discriminant analyses enabled us to discriminate four groups of athletes according to their performance considering several physical variables rather than just one ( i.e. , a multivariate approach ) . using this method ,
players are assigned to clusters by physical variables that identify their strengths and weaknesses . as it can be seen in figure 2
, players assigned to cluster 3 were less physically fit and required improvement in the horizontal ( e.g. , st10 and st20 ) and cross - sectional dimensions ( e.g. , 1rmest and brv ) . to train the specific needs of this group , technique and maximum strength sessions
could be implemented in their training schedule . whereas , players assigned to cluster 1 needed enhancement in the cross - sectional dimension of the upper trunk only ( i.e. , ppeakmp and pmppmp ) .
training sessions based on high execution velocity would be a good choice for this cluster group to improve the variables related to muscular power .
therefore , the cluster analysis allows coaches to group training sessions ensuring players will develop specific skills and abilities to enhance their physical performance parameters .
finally , our discriminant analysis assigned greater relative weight to some of the performance variables assessed compared to others . based on this type of objective quantifiable information , training sessions could be designed to improve performance in these specific physical measures . | abstractthe aim of the present study was to identify different cluster groups of handball players according to their physical performance level assessed in a series of physical assessments , which could then be used to design a training program based on individual strengths and weaknesses , and to determine which of these variables best identified elite performance in a group of under-19 [ u19 ] national level handball players .
players of the u19 national handball team ( n=16 ) performed a set of tests to determine : 10 m ( st10 ) and 20 m ( st20 ) sprint time , ball release velocity ( brv ) , countermovement jump ( cmj ) height and squat jump ( sj ) height .
all players also performed an incremental - load bench press test to determine the 1 repetition maximum ( 1rmest ) , the load corresponding to maximum mean power ( loadmp ) , the mean propulsive phase power at loadmp ( pmppmp ) and the peak power at loadmp ( ppeakmp ) .
cluster analyses of the test results generated four groupings of players . the variables best able to discriminate physical performance were brv , st20 , 1rmest , ppeakmp and pmppmp .
these variables could help coaches identify talent or monitor the physical performance of athletes in their team .
each cluster of players has a particular weakness related to physical performance and therefore , the cluster results can be applied to a specific training programmed based on individual needs . |
this safe and inexpensive 4-aminoquinoline compound accumulates inside the digestive vacuole of the infected red blood cell .
chloroquine resistance ( cqr ) was first reported in southeast asia and south america and has now spread to the vast majority of malaria - endemic countries .
antimalarial drug resistance is a major public health problem which hinders the control of malaria . in india resistance of plasmodium falciparum to chloroquine
was first reported in the year 1973 from diphu of the karbi anglong district in assam state .
various in vitro sensitivity test systems have been developed and applied to sensitivity monitoring of p. falciparum in endemic areas .
the most commonly used methods are in vitro tests based on the measurement of the effect of drugs on the growth and development of malaria parasites , that is , schizont maturation or growth inhibition [ 2 , 3 ] , incorporation of radiolabeled precursors , enzymatic activity of parasite lactate dehydrogenase ( pldh ) , or histidine - rich protein ii ( hrp ii ) .
the in vitro sensitivity test based on the standard micro - technique recommended by the world health organization using the schizont maturation inhibition test has been applied successfully in most of the highly multi - drug - resistant areas worldwide .
the study was done to determine the prevalence of p. falciparum infection in aligarh , a north indian district and to test the chloroquine sensitivity using mark iii who sensitivity plates .
the study was conducted in aligarh , a district of north india , where there is high prevalence of p. falciparum species .
the study was conducted from february 2010 to december 2011 , but majority of cases ( > 90% ) were detected in months of july to october in both years .
a total of 5653 patients , who presented with fever , chills , rigors , or any pyrexia of unknown origin were screened for malarial infection by using different diagnostic tests like peripheral blood smear examination and quantitative buffy coat assay and by various antigen detection assays .
the drug sensitivity was done against chloroquine as it is the most commonly used antimalarial agent in out - patient departments for uncomplicated malarial infection .
we also want to study chloroquine sensitivity because of its improper prescription and dosing in fever of unknown origin in this area by unqualified medical practitioners . 5 ml of blood was collected by venipuncture taking all sterile precaution in heparinized tube .
both thick and thin smears were examined by staining with giemsa stained using standard protocols .
parasitemia was determined by counting 200 wbcs on thick films and expressed as parasites per microliter of blood .
a total of 200 oil emergence fields were examined before the smear is labeled negative or positive .
the in vitro cultivation of p. falciparum isolates followed a modification of the standard culture techniques [ 7 , 8 ] . the culture medium consisted of rpmi 1640 ( sigma aldrich ) ,
2 g glucose , and 40 g / ml gentamycin sulphate with supplemented 10% ab+ serum .
culture medium was sterilised by filtration through a millipore filter of 0.22 m porosity and ph was adjusted to 7.4 by the addition of 4.2 ml of sterile 5% sodium bicarbonate . the drug sensitivity ( based on the m - iii methods )
can be determined in vitro in p. falciparum culture by using standard 96-well microtitre plates ( who plates ) , predosed of the test drug .
h 0 , 1 , 2 , 4 , 8 , 16 , 32 , and 64 pmol .
blood medium mixture ( bmm ) was prepared by gently shaking the tube to mix the blood and medium .
all the wells of appropriate column were dosed with 50 l of the blood medium mixture ( 1 : 9 ) . the plate was put in a candle jar and placed in the incubator set at 37.5c for 2430 hrs , depending upon development stage of the parasite .
after 24-hour incubation , a thin smear was prepared from the control well to see the mature schizonts , and if more than 10% schizonts were seen , it was considered to be valid ; thick smears were prepared from each well by discarding the excess media with a micropipette .
the mean number of schizonts counts per well was fed directly into nonlinear regression software , h n - non - lin v1.1 , specific for malaria in vitro drug sensitivity test .
individual dose response curves were generated and their ec 50 , ec 90 , and ec 99 values determined .
the geometric means and 95% confidence intervals ( cis ) of ec values were estimated in spss ( spss inc .
, usa ) . out of 5653 patients who presented with pyrexia of unknown origin or pyrexia associated with symptoms suggestive of malarial infection , only 435 patients had p. falciparum infection .
fever was present in all patients ( 100% ) ; chills ( 85.51 ) , rigors ( 71.26% ) , and altered level of consciousness ( 60.74% ) were the common features with which p. falciparum positive cases presented ( table 1 ) .
four hundred and thirty - five p. falciparum - positive blood samples were cultured ; 108 cultures were positive ; from all these isolates when subjected to chloroquine sensitivity , 26 isolates were found to be resistant to chloroquine , that is , schizont maturation at 8 pmols or more .
isolates showed schizont maturation up to 32 pmols or more ( figure 1 ) .
the mean effective concentrations of chloroquine ec 50 = 6.07 nm / liter , ec 90 = 33.33 nm / liter , ec 95 = 43.44 nm / liter , and ec 99 = 47.40 nm / liter , which shows decreased sensitivity of p. falciparum isolates to chloroquine ( table 2 ) .
the major obstacle in malaria treatment and control is impeded by the drug - resistant parasites , particularly plasmodium falciparum , which have disseminated and enhanced mortality .
chloroquine drug pressure remains high in india as it has been used since decades as first - line drug in malaria therapy both as self - treatment at home and in health - care facilities shah et al .
analyzed 337 studies and investigated chloroquine efficacy in 17189 patients . the number of studies and proportion of failures varied between regions and in states within a region .
the median proportion of chloroquine failure was 35.1% ( iqr 13.058.2 ) in studies with a 28-day followup .
studies with 7-day followup , which largely detected early treatment failures , were phased out from 2000 and the last 7-day study was done in 2003 . the proportion of failures detected is higher in 28-day follow - up studies than in 7-day follow - up studies done in the same areas , because late treatment failures are detected in 28-day follow - up studies .
studies done between 1978 and 2007 show an increasing proportion of failures to chloroquine over time .
when routine monitoring of drug resistance began in 1978 - 1979 , two of 17 studies exceeded the 10% threshold used in india to switch an area to the second - line treatment . in 2006 - 2007 ,
the proportion of chloroquine studies exceeding 10% treatment failure increased to 35 of 40 studies .
drug - efficacy studies of chloroquine , with at least 30 patients in any follow - up period , have exceeded 10% treatment failure in 115 districts .
the remaining states and territories have low incidence of p. falciparum , or have not done any trial of antimalarial drug resistance .
only 31% cases have given adequate culture results due to low parasitemia , previous history of inadequate treatment by quacks .
this is a tertiary care hospital where most of the patients come after getting treatment from one or more sources .
most of the studies done in india mainly determined the in vivo resistance patterns of chloroquine .
there is paucity of data from india regarding in vitro drug resistance pattern in india . in our study
when chloroquine was tested against p. falciparum isolates , high level of resistance was noticed by in vitro mark iii sensitivity assay 53.76% of isolates resistant to 8 pmols of chloroquine which is the who cut - off value for sensitivity .
the level of resistance in this part of india is so high that 10.75% strains were resistant to even 32 pmols of drug .
one of the major objectives of the study is to determine effective concentration values ( ec values ) of chloroquine .
our results showed that mean ec5o value of sensitive strains was 1.30 and for resistant 6.07 ( nmol / liter ) , ec 90 , ec 95 , and ec 99 were found to be 3.94 nm / liter and 33.33 nm / liter , 5.56 nm / liter and 43.44 nm / liter , 6.17 nm / liter , and 47.40 nm / liter for sensitive and resistant isolates , respectively .
these results indicate high level of resistance of p. falciparum to chloroquine among the isolates tested , and this is confirmed by high ec values which are excessively higher than effective therapeutic concentration .
these values are significantly higher than those reported from india and other countries [ 13 , 14 ] .
this high - level resistance to chloroquine in our patients may be due to previous exposure to improper therapeutic regimes , over - the - counter availability of drug , and high drug pressure by improper prescribing habits of private practitioners .
so this high level of resistance should be monitored regularly to prevent further spread of resistant strains .
the use of combination therapy should be advocated to prevent the drug resistance in malaria - endemic areas . | chloroquine ( cq ) has been the mainstay of treatment of malaria for decades .
this cost - effective and safe drug has become ineffective for treatment
of falciparum malaria in many parts of the world due to development of resistance by the parasite .
in addition cq is not gametocytocidal for p. falciparum and thus can not block transmission .
the extent of problem of chloroquine resistance in
p. falciparum is increasing every year .
the study was done in period of 2 years .
a total of 5653 specimens were examined for malarial infection by employing different diagnostic modalities .
four hundred and thirty - five were found to be positive for p. falciparum by using different diagnostic techniques .
all positive specimens were cultured on rpmi 1640 medium ; only 108 were found to be culture positive .
sensitivity of isolates to chloroquine was done using mark iii who sensitivity plates .
the prevalence of malaria infection was found 9.54% in 2010 .
there were schizont formation at 8 pmol / liter or more of chloroquine concentration in 26 isolates .
the emergence of chloroquine ( cq ) resistance pattern
in aligarh isolates increases .
antimalarial agents should be used with caution ; monotherapies should be avoided . |
a 69-year - old male complained of a 4 month history of a progressively enlarging , painless right groin swelling during routine urological review .
he had been diagnosed with locally advanced prostate cancer 2 years previously , for which he had received radical radiotherapy ( 74gy/37 fractions ) and was on maintenance androgen deprivation therapy .
contrast computed tomography of abdomen and pelvis showed a non - obstructed right inguinal hernia containing a contrast filled loop of small bowel ( figure 1 ) .
intra - operatively a solid mass involving the spermatic cord and extending into the proximal scrotum was seen .
pathology revealed a right paratesticular sarcoma , of the leiomyosarcoma type involving the spermatic cord ( figure 2 ) .
figure 1transverse section of a contrast enhanced computed tomographic scan of the abdomen and pelvis showing a right inguinoscrotal mass .
transverse section of a contrast enhanced computed tomographic scan of the abdomen and pelvis showing a right inguinoscrotal mass .
figure 2histologically the tumor appears as malignant smooth muscle spindle cells with typical cigar - shaped nuclei , which stain to alpha actin , desmin , and vimentin antibodies .
histologically the tumor appears as malignant smooth muscle spindle cells with typical cigar - shaped nuclei , which stain to alpha actin , desmin , and vimentin antibodies .
soft - tissue sarcomas of the genitourinary tract are uncommon , accounting for only 12% of urological malignancies , and 2.1% of soft - tissue sarcomas in general .
more specifically para - testicular sarcoma ; sarcomas of the spermatic cord are extremely rare .
leiomyo - sarcoma is the commonest type of paratesticular sarcoma . only around 110 cases of leiomyosarcoma of the spermatic cord
histologically , the most common spermatic cord type arises from undifferentiated mesenchymal cells of the cremasteric muscle and vas deferens .
the less frequent epididymal form arises from the smooth muscle surrounding the basement membrane of the epididymal canal .
leiomyosarcoma may be divided anatomically into deep soft - tissue , cutaneous - subcutaneous , and vascular .
the american joint committee on cancer ( ajcc ) classifies spermatic cord leiomyosarcoma as deep tissue .
nodal spread can involve external iliac , hypogastric , common iliac and para - aortic nodes .
the vas deferens can act as a conduit for local spread , which may involve the scrotum , inguinal canal or pelvis .
clinical presentation is typically with a painless , firm , progressively enlarging , intra - scrotal mass which is extra - testicular .
the differential diagnosis includes inguinoscrotal hernia , hydrocoele , testicular malignancy and other rarer tumors including benign leiomyoma , fibrous mesothelioma , various benign fibrous tumors and pseudotumors , and fibromatosis .
the exact degree to which surrounding soft tissues should be resected and the optimal margin measurements remain controversial . in 27% of cases , which were completely excised ,
nodal involvement appears to be more commonly associated with paratesticular sarcoma than other soft tissue sarcomas . in two separate studies catton and colleagues have shown nodal failure rates varying between 14% ( 2 of 14 patients ) and 28% ( 6 of 21 patients ) . however , the literature has reported rates of up to 29% . due to this high incidence of nodal recurrence , both prophylactic retroperitoneal lymph node dissection and adjunctive nodal radiotherapy have been used .
to date there are no reports of an improvement in survival with adjunctive nodal dissection .
local recurrence rates of 27% have been reported with wide excision alone , although the study population involved was small .
the median follow - up for each was 4.2 years , with the exception of the study by fagundes et al .
it is of note that , in this study , median follow - up for non - irradiated and irradiated groups differed , being 123 and 63 months respectively .
this may have lead to an artificially increased rate of recurrence in the non - irradiated group .
local recurrence has been described as the achilles heel of sarcoma management , and spermatic cord sarcomas are no exception .
sarcomas of all grades have a tendency to infiltrate local tissues , leading to morbidity from local disease progression , and a higher rate of metastasis and disease - specific mortality .
in one study , which looked at 102 patients with primary disease of the genitourinary tract alone , 33 developed local recurrence ( median time to local recurrence being 10.6 years ) with 20 sarcoma - specific deaths .
the risk associated with sarcomas can be reduced with adjunctive radiotherapy , or adjunctive surgery as required . with the exception of the dotan et al .
's study , previous attempts at examining urological sarcomas with regards to optimal management have been hindered by small cohort numbers , and the rarity of the condition .
however , prolonged disease - specific survival has consistently been shown to be associated with tumor histology , size ( < 5 cm ) , grade , achieving complete resection , paratesticular or bladder tumor site and lack of metastases at presentation .
the largest study of genitourinary sarcomas to date looked at 131 patients ; 57 having a para - testicular sarcoma .
51% of the paratesticular patients were disease - free at the median follow - up time of 4.2 years , with 3% still showing evidence of disease , and 39% having died of the disease in the interim .
adjunctive radio- and chemotherapy were not consistently used in this retrospective study population , and therefore their effects were not considered .
this is unfortunate , as it remains to be conclusively proven that adjuvant radiation or chemotherapy is effective in para - testicular sarcomas specifically .
the standard management of wide excision with adjunctive radio - therapy is based on the success of adjuvant radiotherapy in the management of sarcomas in general .
evidence for the routine use of adjuvant chemotherapy for adult high grade sarcoma is controversial . in 1997
the sarcoma meta - analysis collaboration showed significant relapse free survival rates ( median follow - up 9.4 years ) with adjuvant doxorubicin in soft - tissue sarcomas ( n=1568 ) .
randomized control trial to date failed to show an improvement with chemotherapy ( n=351 ) .
phase i and ii trials showed promising preliminary results with objective response rates between 3.7% and 17.1% .
as the majority of studies into paratesticular sarcomas are retrospective by necessity , the inconsistency of management of the patients involved makes a comparison of therapies difficult . | soft - tissue sarcomas of the genitourinary tract account for only 12% of urological malignancies and 2.1% of soft - tissue sarcomas in general .
a 69-year - old male complained of a 4 month history of a painless right groin swelling during routine urological review for prostate cancer follow - up .
clinical examination revealed a non - tender , firm right inguinoscrotal mass .
there was no discernible cough impulse .
computed tomography of abdomen and pelvis showed a non - obstructed right inguinal hernia . during elective hernia repair a solid mass involving the spermatic cord and extending into the proximal scrotum was seen .
the mass was widely resected and a right orchidectomy was performed .
pathology revealed a paratesticular sarcoma .
he proceeded to receive adjuvant radiotherapy . only around 110 cases of leiomyosarcoma of the spermatic cord
have been described in the literature .
they commonly present as painless swellings in the groin .
the majority of diagnoses are made on histology . |
chronic intestinal pseudo - obstruction ( cipo ) is a high - morbidity syndrome that develops as a consequence of altered intestinal motility , which results in clinical manifestations that resemble intestinal obstruction but in the absence of any physical obstructive process .
cipo is one of the most important causes of chronic intestinal failure both in pediatric ( 15% ) and adult cases ( 20% ) , since affected individuals are often unable to maintain normal body weight and/or normal oral nutrition , .
the severity of the clinical picture , generally characterized by disabling digestive symptoms even between sub - occlusive episodes , contributes to deterioration of quality of life of the patients .
furthermore , cipo often passes unrecognized for long periods of time , so that patients almost invariably undergo repeated , unnecessary and potentially invasive surgical procedures .
fabry disease ( fd ) is an x - linked lysosomal storage disease characterized by the dysfunction of multiple systems , including significant gastrointestinal ( gi ) involvement such as diarrhea , abdominal pain , early satiety and nausea .
the deficiency in lysosomal -gal a causes accumulation of globotriaosylceramide ( gl-3 ) within the lysosomes of multiple cell types throughout the body . this accumulation results in inflammation , ischemia , hypertrophy , and the development of fibrosis ultimately resulting in cellular damage and progressive organ dysfunction , . although fd was thought to be rare , only affecting 1 in 40,000 ; recent newborn screening data show it to be much more common , affecting up to 1 in 34004000 newborns .
we report the occurrence of cipo in two unrelated patients with fabry disease and the possible explanation of a direct relation of these two disorders .
case 1 : 62 years old female with classic phenotype of fd ( mutation : a292 t ) . at her initial visit for fd at the age of 59 ,
the patient described isolated bloating and sporadic diarrhea with intermittent abdominal colic pain since the age of 18 .
medical records showed at least 4 hospitalizations for intestinal pseudo - obstruction during the last 20 years .
she had echocardiographic evidence of left ventricular hypertrophy ( lvh ) and had a history of arrhythmias .
she presented with a 5-day history of abdominal pain , primarily in the right lower quadrant and associated with intermittent fevers .
abdominal ct reported distended bowel loops , thickening of the proximal colon wall and free fluid in adjoining fascias .
the following day , physical exam revealed an absence of abdominal sounds and progressively worsening pain .
the patient underwent abdominal surgery with right colonic resection and placement of colostomy due a suspicion of tumoral obstruction .
pathological findings in h&e stained paraffin sections demonstrated enlarged ganglion cells with foamy cytoplasm present within meissner 's plexus of the submucosa present in the ileocecal valve sample .
the foamy appearance likely represented the presence of gl-3 accumulation , lost in the paraffin processing .
epoxy resin sections taken from this same area show enlarged ganglion cells filled with zebra bodies , further suggesting the accumulation of gl-3 .
ganglion cells of auerbach 's plexus present between the two muscular layers of the muscularis propria were also engorged with gl-3 accumulation ( fig .
substrate accumulation was also present in the smooth muscle cells of small arteries present in the submucosa ( fig .
1a : ganglion cells of auerbach 's plexus : ganglion cells appear markedly foamy due to the accumulation of gl-3 ( paraffin section , h&e , 600 ) .
( hrlm , 1 m epoxy resin , 1:1 richardson 's stain , 1000 ).fig .
1 a : ganglion cells of auerbach 's plexus : ganglion cells appear markedly foamy due to the accumulation of gl-3 ( paraffin section , h&e , 600 ) .
( hrlm , 1 m epoxy resin , 1:1 richardson 's stain , 1000 ) .
case 2 : 37 years old male patient was diagnosed with classic fd ( mutation : d155h ) following a renal biopsy for proteinuria study .
the patient had other classic symptoms of fd including chronic abdominal pain and episodes of diarrhea during childhood , neuropathic pain in the hands and feet associated with fever and exercise , angiokeratomas and cornea verticillata .
furthermore , echocardiogram showed mild lvh and brain mri showed small periventricular ischemic lesions . at the age of 32
, the patient was hospitalized due abdominal pain and diverticulitis ; after 2 days of conservative treatment he made a full recovery . at the age of 38 , the patient developed fever and progressive left , lower - quadrant abdominal pain .
after 3 days of conservative treatment , physical exam revealed severe abdominal distention , absence of abdominal sounds and progressively worsening pain .
abdominal surgery showed sigmoid colon distention and a delustered appearance ; bowel resection with placement of colostomy was performed .
pathological examination of h&e stained paraffin revealed neither obstructive process nor inflammatory infiltration . enlarged and vacuolated neurons in the meissner 's and auerbach 's plexuses ( fig .
2a ) and intracellular inclusions in the smooth muscle cells of small arteries were present in the submucosa .
b : vascular arterial lumen narrowing due smooth muscle cells vacuolated cytoplasm . ( paraffin section , h&e , 600).fig .
2 a : vacuolated cytoplasm of submucosal and myenteric plexus colon cells . ( paraffin section , h&e , 600 ) .
b : vascular arterial lumen narrowing due smooth muscle cells vacuolated cytoplasm . ( paraffin section , h&e , 600 ) .
cipo may be primary , when it exclusively involves intestinal smooth muscle or enteric neural plexus , or secondary to other conditions .
the histopathologic presentation of cipo can be divided in neuropathic , mesenchymopathic and myopathic forms based on abnormalities affecting the integrity of nerve pathways supplying the gut ( either intrinsic or extrinsic ) , interstitial cells of cajal ( icc ) or smooth muscle cells , respectively .
neuropathic , mesenchymopathic and myopathic changes may contribute to gut dysmotility either individually or in combination ( e.g. neuro - myopathies or neuro - icc alterations ) .table 1causes of chronic intestinal pseudo - obstruction syndrome.table 1myopathicneuropathicmesenchymopathicvisceralmitochondrialcentral:parkinson diseasemultiple system atrophystrokeencephalitistumorloss of interstitial cells of cajalprimary:absence of or selective decrease in smooth - muscle alpha - actinfamilial or sporadic visceral myopathymyopathy from abnormal gut morphogenesisautoimmune leiomyositisdiseases from defective genes coding for proteins indirectly related to oxidative phosphorylationperipheral:diabetic neuropathyneuropathies potentially involving the enteric nervous system : amyloidosis hirschsprung chagas paraneoplastic syndromes autoimmune diseasessecondary:lupus , polymyositisamyloidosis ceroidosis ( vitamin e deficiency)progressive muscular dystrophy,drugs ( neuroleptics)diseases from genetically - induced mitochondrial dna stability disturbancediseases from defective nuclear genes coding for crm enzyme complex proteins causes of chronic intestinal pseudo - obstruction syndrome .
multiple system atrophy absence of or selective decrease in smooth - muscle alpha - actin familial or sporadic visceral myopathy myopathy from abnormal gut morphogenesis autoimmune leiomyositis neuropathies potentially involving the enteric nervous system : paraneoplastic syndromes amyloidosis ceroidosis ( vitamin e deficiency ) progressive muscular dystrophy , in fd , gastrointestinal symptoms occur in approximately 70% of male patients , , but the frequency ranges from 19% to 69% in different series , . in heterozygous patients ( females )
almost half of them may experience gi symptoms and some of those symptoms , such as constipation , are reported almost twice as often by female patients as by male patients .
significant gi involvement has also been observed in children and can progress in severity with age . in a systematic review ,
laney and colleagues observed that gi symptoms even presented in very young children , aged 14 years old , most commonly complaining of abdominal pain .
the symptoms present soon after the development of acroparesthesias and can be the initial symptom of fd in up to 20% of patients .
abdominal pain is described as cramping mid - abdominal discomfort , frequently worsened with meals and increased stress .
the possible local ischemia due endothelial deposits of gl-3 has led to the suspicion of
. the diarrhea can be intake - triggered , frequently associated with significant urgency and frequency , and occurring up to 15 times daily .
, colonic dysmotility can lead to the pseudo - obstruction syndrome , simulating intestinal necrosis .
that is why up to this date colostomy has been performed in some cases , , even for children with fd without cardiac , renal or cerebrovascular compromise .
intestinal perforation , secondary to diverticular disease , has been repeatedly described in the literature .
the location of diverticula has been described as occurring at the duodenal , jejunal , and colonic levels , , .
neuropathic pain ( acroparesthesias ) and gi involvement may be explained due a multifactorial cascade .
substrate deposits of gl-3 in autonomic ganglia ( dorsal root ganglia -drg- and myenteric and submucosal ganglia ) may be the cellular alteration which results in the appearance of symptoms , , , , .
intestinal ischaemia due myenteric vessel occlusion could also be correlated with the small fiber neuropathy as a result of vasa nervorum obliteration .
animal models with fd showed mechanical hypersensitivity in the von- frey test and mechanical allodynia when compared with controls , .
the same studies demonstrated that tissue involvement in drg and small nerve fiber loss are linked with upregulation of sodium channels type 1.8 ( nav1.8 ) and transient receptor potential vanilloid type 1 ( trpv1 ) .
the upregulation of these channels in drg neurons causes an early depolarization of the resting potential , reduced threshold and increased frequency for evoked firing in these neurons , and increased fraction of drg neurons that fire spontaneously .
the gi manifestations reported by patients with fd might be explained by the effect of the gl-3 deposits on visceral afferents that are known to express nav1.8 and trvp1 channels .
a specific role for nav1.8 in regulating gastrointestinal function is supported by experimental evidence showing that this channel ( as well as nav1.9 also ) is important for responses to mechanical stimulation and mechanical hypersensitivity of visceral afferents innervating the colon when challenged by an inflammatory mediators or food intake .
enzyme replacement therapy ( ert ) should be initiated in all affected patients with fd as soon as clinical signs and symptoms are observed .
early treatment of asymptomatic or mildly symptomatic patients may be justified in order to prevent disease progression .
this may be explained by a decrease of gl-3 accumulation in the endothelium of the intestinal vessels and by improved mesenteric circulation .
ert is presently available in the form of agalsidase alfa ( replagal , shire hgt , inc . , cambridge , ma , usa ) and agalsidase beta ( fabrazyme , sanofi genzyme , cambridge , ma , usa ) , .
agalsidase alfa is given at 0.2 mg / kg body weight every other week by intravenous ( iv ) infusion and is approved in many countries throughout the world , though not by the usa food and drug administration .
agalsidase beta is administered at 1.0 mg / kg body weight once every 2 weeks as an iv infusion and is approved in europe , the usa and many other countries .
there are no adequately powered head - to head studies that have compared the long - term effectiveness of the two enzyme preparations on clinical outcomes in well - characterized , phenotypically homogenous populations but evidence suggests that a higher agalsidase dose may be of clinical benefit in reducing gl-3 accumulation , . because of the shortage of agalsidase - beta in 2009 , many patients with fd were treated with lower doses or were switched from agalsidase beta to agalsidase alfa .
one observational study assessed end - organ damage and clinical symptoms during dose reduction or switch from agalsidase beta to alfa .
after 1 year , severity score index and frequencies of pain attacks , chronic pain , gastrointestinal pain , and diarrhea increased significantly in the dose - reduction and switch groups .
because of the non - specific nature of the gi symptoms , diagnosis of fd is often delayed for several years .
gastrointestinal involvement is often misdiagnosed ( usually as irritable bowel disease , visceral myopathy or inflammatory bowel disease ) or under - reported .
the other authors declare that they have no competing interests in relation to this work .
this research did not receive any specific grant from funding agencies in the public , commercial , or not - for - profit sectors .
all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation of laboratorio de neuroqumica dr . | chronic intestinal pseudo - obstruction results in clinical manifestations that resemble intestinal obstruction but in the absence of any physical obstructive process .
fabry disease is an x - linked lysosomal storage disease characterized by the dysfunction of multiple systems , including significant gastrointestinal involvement .
we report the occurrence of chronic intestinal pseudo - obstruction in two unrelated patients with fabry disease and the possible explanation of a direct relation of these two disorders . in fabry disease ,
gastrointestinal symptoms occur in approximately 70% of male patients , but the frequency ranges from 19% to 69% in different series . in some patients , colonic dysmotility due glycolipid deposition in autonomic plexus and ganglia can lead to the pseudo - obstruction syndrome , simulating intestinal necrosis .
that is why up to this date colostomy has been performed in some cases , even for children with fd without cardiac , renal or cerebrovascular compromise .
early treatment with enzyme replacement therapy in asymptomatic or mildly symptomatic patients may be justified in order to prevent disease progression .
several studies have demonstrated that enzyme replacement therapy alleviates gi manifestations . because of the non - specific nature of the gastrointestinal symptoms , diagnosis of fabry disease is often delayed for several years .
gastrointestinal involvement is often misdiagnosed or under - reported .
it is therefore very important to consider fabry disease in the differential diagnosis of chronic intestinal pseudo - obstruction . |
there have been conflicting reports on the role of ovary ( estrogen ) and testis ( testosterone ) in growth and cholesterol metabolism .
our recent study ( lee et al . , 2008 ) demonstrated that a lower growth rate in female rats was due to estrogen , which induced secretion of the stress hormone cortisol .
similarly , other studies ( burgess & handa , 1992 ; lesniewska et al . , 1990 ) also showed gender differences in cortisol secretion , indicating that estrogen elevates basal levels of cortisol , corticosterone and adrenocorticotropin , and their responses to various stimuli in rats .
short - term estradiol treatment to young men increased cortisol and norepinephrine concentrations in the saliva following stress over a placebo control ( kirschbaum et al . , 1996 ) . in the framingham study
, the risk of cardiovascular disease was shown to be significantly increased in women who had taken estrogen ( wilson et al . , 1985 ) .
( 1996 ) reported that estrogen administration resulted in an enhanced cardiovascular response to mental stress in young menstruating women .
in contrast , estrogen has long been known to exert cardioprotective effect ( barrett - connor & bush , 1991 ) , although the precise mechanism underlying its benefits is unknown ( sudhir et al . , 1997 ) .
antioxidant activity of estrogen has been suggested as its role in cardioprotection ( walsh et al . , 1999 ) , but santanam et al .
( 1998 ) found no inhibitory action of estrogen on ldl oxidation at physiological concentrations .
2008 ) showed a much higher plasma cholesterol level in female than in male rats when they were fed a hypercholesterolemic diet ( cd ) .
naito et al . ( 1995 ) proposed that the effect of sex hormones on lipid metabolism is not likely to account for the sex difference in cardiovascular disease .
furthermore , in the 1995 ancel keys lecture , barrett - connor ( 1997 ) indicated that plasma estrogen levels do not explain coronary heart disease ( chd ) risk in either sex . in the present study , we conducted two experiments to determine the effect of gender and gonadectomy on growth , feed efficiency and plasma cholesterol levels , using pigs as a model animal to confirm the findings of our previous study done with rats ( lee et al . , 2008 ) .
in experiment 1 conducted to assess the effect of gonadectomy ( gdx ) on growth and feed efficiency in female pigs , five sham - operated intact and five gdx landrace female pigs ( 26 kg ) were allowed to have free access to water and feed until they reached market weight ( approximately 100 kg ) .
body weight and feed consumption were recorded biweekly , and daily body weight gain , daily feed intake and feed efficiency ( gain / feed ) were calculated during the feeding period . in experiment 2 conducted to determine the effect of gender and gdx on plasma cholesterol levels in pigs , 10 male ( 26 kg ) and 10 female ( 26 kg ) landrace pigs
were used ; five male and five female pigs were assigned to sham - operated intact or gdx .
pigs were allowed to have free access to water and diet without added cholesterol until they were 6 months old ( male 104 and female 98 kg ) and thereafter a hypercholesterolemic diet ( table 1 ) containing 0.5% cholesterol and 0.1% cholate for 10 days .
all animal management and sampling procedures were in accordance with the guide for the care and use of agricultural animals in agricultural research and teaching ( consortium , 1988 ) .
testicles were removed through a small incision in the tip of the scrotum after ligation of the spermatic cords .
ovariectomy was done through a dorsal paramedial incision at the level of the lower pole of the kidneys .
the surgery on pigs used in both experiments was done when they were 10 days old . at the end of feeding period ,
1 ) , and from the pigs used in experiment 2 , blood samples were collected into vacutainer tubes containing edta before and after a 10-day feeding period of the hypercholesterolemic diet . plasma was prepared from the blood samples by centrifugation , and stored at -20 for later analysis . for pigs used in experiment 1 ,
the average value of measurements at 11 thoracic and 1 lumbar vertebra was used as the back fat thickness .
total and high - density lipoprotein ( hdl ) cholesterol concentrations in plasma were determined using commercial assay kits ( international reagent corp . ,
low - density lipoprotein ( ldl ) plus very - low - density lipoprotein ( vldl ) cholesterol concentration was calculated by subtracting hdl cholesterol from the total .
the student t - test was used to assess the effect of ovariectomy on pig performance in experiment 1 .
data from experiment 2 were analyzed by the two - way analysis of variance ( sas , 1988 ) . in the analysis of variance ,
when the f - value in the analysis of variance was significant , the duncan 's multiple range test was used to compare individual means .
in experiment 1 conducted to assess the effect of gonadectomy ( gdx ) on growth and feed efficiency in female pigs , five sham - operated intact and five gdx landrace female pigs ( 26 kg ) were allowed to have free access to water and feed until they reached market weight ( approximately 100 kg ) .
body weight and feed consumption were recorded biweekly , and daily body weight gain , daily feed intake and feed efficiency ( gain / feed ) were calculated during the feeding period . in experiment 2 conducted to determine the effect of gender and gdx on plasma cholesterol levels in pigs , 10 male ( 26 kg ) and 10 female ( 26 kg ) landrace pigs
were used ; five male and five female pigs were assigned to sham - operated intact or gdx .
pigs were allowed to have free access to water and diet without added cholesterol until they were 6 months old ( male 104 and female 98 kg ) and thereafter a hypercholesterolemic diet ( table 1 ) containing 0.5% cholesterol and 0.1% cholate for 10 days .
all animal management and sampling procedures were in accordance with the guide for the care and use of agricultural animals in agricultural research and teaching ( consortium , 1988 ) .
testicles were removed through a small incision in the tip of the scrotum after ligation of the spermatic cords .
ovariectomy was done through a dorsal paramedial incision at the level of the lower pole of the kidneys .
the surgery on pigs used in both experiments was done when they were 10 days old . at the end of feeding period
1 ) , and from the pigs used in experiment 2 , blood samples were collected into vacutainer tubes containing edta before and after a 10-day feeding period of the hypercholesterolemic diet .
plasma was prepared from the blood samples by centrifugation , and stored at -20 for later analysis . for pigs used in experiment 1 ,
the average value of measurements at 11 thoracic and 1 lumbar vertebra was used as the back fat thickness .
total and high - density lipoprotein ( hdl ) cholesterol concentrations in plasma were determined using commercial assay kits ( international reagent corp . ,
low - density lipoprotein ( ldl ) plus very - low - density lipoprotein ( vldl ) cholesterol concentration was calculated by subtracting hdl cholesterol from the total .
the student t - test was used to assess the effect of ovariectomy on pig performance in experiment 1 .
data from experiment 2 were analyzed by the two - way analysis of variance ( sas , 1988 ) . in the analysis of variance ,
when the f - value in the analysis of variance was significant , the duncan 's multiple range test was used to compare individual means .
overiectomy increased average daily gain ( p<0.05 ) , but had no effect ( p>0.5 ) on feed efficiency during the growing - finishing period ( table 2 ) . back
fat thickness and dressing percentage in ovariectomized pigs tended to be higher ( p>0.05 ) than those found in sham - operated intact female pigs ( table 2 ) .
no differences in the plasma total or hdl cholesterol levels and hdl / ldl+vldl ratio were found before feeding the hypercholesterolemic diet between sexs or between intact and gdx ( table 3 ) . however , plasma cholesterol levels in pigs fed the hypercholesterolemic diet for 10 days were much higher in females than in males ( 161 vs 104 mg/100 ml plasma ) .
hdl / ldl+vldl ratio appeared to be higher ( p<0.01 ) in male than in female pigs , and was not influenced by gdx in either sex .
the present pig study data ( table 2 ) support our previous findings of the rat study ( lee et al . , 2008 ) ,
in which depressed growth in female rats compared with male rats was mostly due to their ovarian activity .
estrogen secreted in the ovary was responsible for the growth depression because the injection of estradiol markedly depressed growth in gdx rats of either sex , while enhancing plasma cortisol levels .
this gender difference was abolished by ovariectomy and was reinstated by estradiol administration ( le mevel et al . , 1979 ) .
the pattern of cortisol secretion was also different between sexes in the nonhuman primate macaques , and estradiol implant in castrated male macaques elicited a female pattern of plasma cortisol levels ( norman et al .
, 1992 ; smith & norman , 1987 ) . in healthy men , short - term treatment with estradiol
led to enhanced hypothalamic - pituitary - adrenal ( hpa ) and sympathetic responsiveness to psychological stress , resulting in increased adrenocorticotropic hormone ( acth ) , cortisol and norepinephrine concentrations in the saliva compared to the placebo ( kirschbaum et al . , 1996 ) .
similarly , burgess and handa ( 1992 ) showed that corticosterone and acth levels after a 5-second footshock stress with one mamp were much higher and maintained for a prolonged time in ovx rats when administered with estradiol .
in addition , they found that estrogen treatment resulted in a loss of the glucocorticoid receptor 's ability of autoregulation .
interestingly , estrogen has been reported to have both stimulatory and inhibitory effects on hpa functions , depending on the time after ovariectomy and different doses of estradiol ( luber et al . , 1991 ; redei et al . ,
cortisol , as well as catecholaminergic responses to stress , has also been known to vary with estrous cycle in women ( collins et al . , 1985 ) and in rats ( carey et al . , 1995 ) .
2008 ) and the present pig studies , and others ' ( le mevel et al . , 1979 ; phillips & poolsaguan , 1978 ) clearly indicate that estrogen induces the secretion of hormones that increase with stress , in turn resulting in growth depression .
therefore , ovariectomy may be an economically viable practice to improve growth of female pigs .
studies have shown marked gender difference in plasma cholesterol levels in sprague dawley rats ( lee et al . , 1999 ; lee et al . , 2008 ) and in guinea pigs ( fernandez et al . ,
the present study also showed a gender difference in plasma cholesterol levels in pigs , and gonadectomy of male but not female increased plasma total cholesterol and ldl - c concentrations without decreasing hdl - c when pigs were fed the hypercholesterolemic diet ( table 3 ) .
testosterone replacement therapy in hypogonadal and elderly men showed a beneficial effect on cardiovascular system , decreasing total cholesterol and atherogenic fraction of ldl - c without significant alterations in hdl - c levels or its subfractions hdl2-c and hdl3-c ( zgliczynski et al . , 1996 ) .
( 1997 ) also showed that increased testosterone levels were associated with reduced triacylglycerol and apo b ; total and ldl cholesterol concentrations ; and increased hdl / total cholesterol and hdl2-c / hdl3-c ratios .
hypotestosteronemia was observed in chinese male patients with coronary heart disease ( chd ) , and positive correlation of plasma testosterone level with plasma hdl - c was found but negative correlation with plasma lp(a ) level , suggesting that testosterone had a protective effect against atherosclerosis ( zhao & li , 1998 ) . on the contrary
, several studies have shown that testosterone has an adverse effect on cholesterol metabolism by altering lipoprotein profiles .
total testosterone concentrations and sex hormone - binding globulin ( shbg ) were significantly associated with ldl size in men ( haffner et al . , 1996 ) .
testosterone , per se , when administered at sufficiently high doses and for long durations , can significantly raise serum levels of total cholesterol , triacylglycerol , ldl - c and apo - b , meanwhile lower hdl - c levels in androgenized women , indicating that male predilection for cardiovascular disease may be due to adverse effects of high androgen levels on lipid and lipoprotein profiles ( goh et al . , 1995 ) .
( 1995 ) also showed that serum hdl - c levels were significantly depressed by intramuscular injection of testosterone enanthate ( 200 mg per week for 12 months ) , while plasma total and ldl cholesterol or triacylglycerol levels were not affected .
1997 ) reported that the plasma free testosterone level was associated with lower levels of hdl - c , but total estradiol was related to elevated levels of hdl - c in japanese men in their early fifties , supporting that testosterone may be causally associated with atherosclerosis in men through altered lipoprotein metabolism .
male hamsters fed a hypercholesterolemic diet containing 0.05% cholesterol developed greater elevations in plasma total cholesterol and hdl - c levels , and a higher rate of early aortic atherosclerosis , compared with female hamsters ( wilson et al . , 1999 ) .
further studies appear to be inevitable to clarify the involvement of testosterone in cholesterol metabolism , such as cholesterol synthesis ( e.g. , hmg - coa reductase gene expression ) and absorption , or cholesterol oxidation and excretion ( e.g. , cholesterol 7-hydroxylase gene expression ) .
estrogen 's involvement in lipoprotein metabolism ( e.g. , an increased ratio of hdl to ldl cholesterol ) has been implicated in its role in cardioprotection ( campos et al .
if estrogen plays any role at all in reducing cardiovascular disease in females , it may be through actions on other than controlling blood cholesterol levels .
naito et al . ( 1995 ) indicated that the effect of sex hormones on lipid metabolism is not likely to account for the sex difference in cardiovascular disease because : 1 ) men with premature myocardial infarction was shown to have increased estrogen levels ( phillips , 1976 ) , and 2 ) men who had received high doses of estrogen showed an increased frequency of cardiovascular events ( coronary drug project research group , 1976 ; veterans administration cooperative urological research group , 1967 ) . in conclusion , ovarian activity depresses growth in female pigs and thus ovariectomy ( or inhibition of estrogen secretion or its activity ) can be an economically viable method to improve growth of female pigs .
testis ( testosterone ) is responsible for the lower plasma cholesterol level in male pigs than in female pigs when they were fed hypercholesterolemic diets . | we conducted two studies to determine the effect of gender , gonadectomy ( gdx ) on growth and plasma cholesterol levels in pigs . in experiment 1 , five sham - operated and five gdx female landrace pigs ( 26 kg ) were allowed to have free access to water and feed up to market weight ( approximately 100 kg ) .
body weight and feed consumption were recorded biweekly , and daily body weight gain , daily feed intake and feed efficiency ( gain / feed ) were calculated during the feeding period . in experiment 2 , 10 male ( 26 kg ) and 10 female ( 26 kg ) landrace pigs were used ; five male and five female pigs were assigned to sham - operated or gdx .
pigs were allowed to have free access to water and a diet without added cholesterol ( table 1 ) until they were 6 months old ( male 104 and female 98 kg ) and thereafter they were fed a hypercholesterolemic diet ( table 1 ) containing 0.5% cholesterol and 0.1% cholate for 10 days .
gdx of female pigs increased average daily gain ( p<0.05 ) , compared with their sham - operated counterparts during the growing - finishing period , but had no effect ( p>0.05 ) on feed efficiency .
plasma cholesterol levels in pigs fed a hypercholesterolemic diet for 10 days were much higher ( p<0.05 ) in females than in males ( 161 vs 104 mg/100 ml plasma ) , and were increased by gdx only in male pigs .
hdl - cholesterol / ldl+vldl - cholesterol ratio appeared to be higher in males than in females , and was not influenced by gdx in either sex .
results suggested that the lower growth rate of female pigs than their male counterparts is attributable to the ovarian activity , and the lower plasma cholesterol level in male than in female pigs fed a hypercholesterolemic diet is due to the testicular activity . |
rodents are commonly used for inhalation toxicology studies , but until recently the nasal passages have often been overlooked or only superficially examined .
the rodent nose is a complex organ in which toxicant - induced lesions may vary , depending on the test compound .
a working knowledge of rodent nasal anatomy and histology is essential for the proper evaluation of these responses .
lack of a systematic approach for examining rodent nasal tissue has led to a paucity of information regarding nonneoplastic lesions in the rodent nose .
therefore , slides from the national toxicology program ( ntp ) and the chemical industry institute of toxicology ( ciit ) were examined , and the literature was reviewed to assemble the spectrum of nonneoplastic rodent nasal pathology .
presented are lesions associated with the various types of epithelia lining the rodent nasal cavity plus lesions involving accessory nasal structures . even though there are anatomic and physiologic differences between the rodent and human nose , both rats and mice provide valuable animal models for the study of nasal epithelial toxicity , following administration of chemical compounds.imagesplate 1.plate 2.plate 3.plate 4.plate 5.plate 6.plate 7.plate 8.plate 9.plate 10.plate 11.plate 12.plate 13.plate 14.plate 15.plate 16.plate 17.plate 18.plate 19.plate 20.plate 21.plate 22.plate 23.plate 24.plate 25.plate 26.plate 27.plate 28.plate 29.plate 30.plate 31.plate 32.plate 33.plate 34.plate 35.plate 36.plate 37.plate 38 . | |
radiation pneumonitis is a dose - limiting complication for patients undergoing radiation therapy for lung cancer .
the incidence of grade ii radiation pneumonitis is relatively low for prescribed standard doses up to 65 gy , but dose escalation is likely to increase the incidence of radiation pneumonitis .
the dose parameters like mean lung dose and the volume of lung receiving greater than 20 gy , 25 gy , or 30 gy are used to estimate the incidence of radiation pneumonitis.[24 ] besides the development of radiation pneumonitis , reduction in overall pulmonary function or lung perfusion due to the treatment can be a complication as well .
the amount of pulmonary function loss is especially important for patients , with medically inoperable non - small - cell lung cancer , who often have a reduced lung function before treatment because of chronic obstructive pulmonary disease ( copd ) , intra - thoracic tumor or because they are heavy smokers .
the extent of damage to the lung due to these pre - existent diseases is not always reflected in computed tomography ( ct ) images .
single photon emission computed tomography ( spect ) lung perfusion scans provide additional information in three dimensions about local functionality of lung tissue and might give additional benefit to design the plan that minimizes the complication risk for perfusion damage for an individual patient .
changes in overall lung perfusion correlate with reduction in pulmonary function tests for patients of lung cancer .
marks et al . , suggested that the perfusion weighted dose volume histogram ( where the volume receiving a certain dose is weighted with the average perfusion in that dose - region ) could be a valuable tool in designing the optimal radiotherapy plan .
the purpose of this study was to evaluate the utility of perfusion spect in conventional radiation treatment planning for locally advanced non - small cell lung cancers .
we first evaluated the accuracy of co - registering ct and spect data in phantom using a public domain freeware .
thereafter , co - registered data were used in patients to evaluate the change in dose volume histogram parameters due to information of functional lung ( fl ) .
twelve patients of locally advanced non - small cell lung cancer suitable for radical radiotherapy consented for the study .
the phantom study was performed to assess the accuracy of ct and spect co - registration .
the jaszczak phantom cylinder interior dimension of 8.5 diameter 7.32 heights ( 21.6 cm 18.6 cm ) containing variable dimension cold rods and solid spheres were used in this study .
the phantom was filled with water and labeled with technetium 99 m ( tc ) of 15 mci .
the spect study was carried out in the gamma camera ( sofa vision medical ( smv ) dstxl , ge health care ) with low - energy high resolution collimators .
the spect study was performed with 64 projections , matrix size of 128 128 , and a pixel resolution of 4.6 mm .
the radiotherapy planning ct ( picker pq 5000 ct scanner , philips medical system ) of the phantom performed in the same orientation with the slice thickness 5 mm , matrix size of 512 512 pixels , and 430 mm field of view correspond to a pixel resolution of 0.84 mm .
the spect matrix was re - sampled to 512 512 matrixes as per the ct data and then co - registered using the statistical parametric mapping software ( spm 2 ) developed by department of neurosciences , university of london software in the matlab platform .
the co - registration accuracy between the spect and the ct data was < 2 mm .
radiotherapy planning ct scans were acquired using three ct / spect compatible markers ( multi - modality markers ) on the patient 's chest in the treatment position using a flat board .
lung perfusion spect images were acquired in a separate session with the same markers at the same position tattooed during ct images .
lung perfusion scans were acquired after an intravenous injection of 200 mbq of tc labeled macro - aggregated albumin using gamma camera on a flat couch , in free breathing .
thereafter both ct and spect images were co - registered using spm 2 software ( statistical parametric mapping ) under matlab 7 platform .
the registration method used here is based on rigid - body model work by collignon et al
. a rigid - body 3d transformation can be parameterized by three scalar translations and rotations matrix perpendicular to each other .
the ct data were kept as a reference and the transformed spect image was processed and re - sliced to a series of registered images so that they match the first image selected voxel - for - voxel .
the accuracy of the co - registration was assessed by superposition of the markers in both the images .
the co - registered ct data with spect was opened using mricro software and the region of interest ( roi ) were delineated ( areas of perfused lung ) .
this produced a multi - colored image , which allowed more accurate volume contouring around a chosen color .
the threshold level was adjusted individually for each patient to match the size of the spect image to the lung volumes defined on ct .
the rois were overlaid on the ct and the data set was transferred on to the treatment planning system ( isis 3d , technology diffusion , france ) for designing the beam portals .
a phantom study was performed to validate the accuracy of the registration algorithm [ figures 1 and 2a d ] .
the accuracy of the registration was evaluated by the superposition of the fiducial markers of the single photon emission computed tomography overlaid image on the computed tomography the accuracy of the registration was evaluated using a phantom .
( a ) jaszezak phantom , ( b ) computed tomography ( ct ) image , ( c ) single photon emission computed tomography ( spect ) image , and ( d ) fused ct spect image for treatment planning purposes , the following areas were outlined for each patient on the co - registered images in the planning system ; gross tumor volume , body outline , anatomic whole lung ( wl ) lung volume based on ct images as a single organ excluding gross target volume ( gtv ) , functional whole lung based on lung volumes visible on spect images ( fl ) , right lung and left lung , and normal structures . the clinical target volume was created using a 2 cm uniform margin around the gtv . planning target volume ( ptv )
was created with an additional 1.5 cm margin for beam characteristics , setup uncertainties , and organ motion .
fl outlines drawn using the spect images were validated by a nuclear medicine expert in functional imaging .
conventional treatment plans were designed to deliver 60 gy to the ptv with an intent to minimize the dose to the ct defined whole lung ( anatomical wl ) or spect defined whole lung ( fl ) to a dose less than 20 gy or 30 gy .
two treatment plans were created for each patient , one an anatomic plan based on ct scan data alone and the other a functional plan with the incorporation of fl information [ figure 3 a
plan comparison was done using dose volume parameters , v20 ( volume of lung receiving 20 gy ) , v30 ( volume of lung receiving 30 gy ) , and mean lung dose ( mld ) .
for each patient the following data were computed : gtv , whole lung volume ( wlv ) , functional lung volume ( flv ) , ptv95 ( % volume of ptv covered by the 95% isodose ) .
dose volume parameters recorded due to ct - based plan were wl_v20 , wl_v30 , and mean lung dose ( wlmld ) . due to spect - based plan
the following data were computed : fl_v20 , fl_v30 , and mean lung dose functional lung mean lung dose ( flmld ) .
comparison of the dose distribution of ( a ) anatomic and ( b ) functional plan along with the respective dose volume histograms ( c and d ) the difference between dose volume parameters for the anatomic and functional plans was compared using bland - altman plots . in bland - altman plots ,
the differences between the methods that were compared were plotted against their means and gave an unbiased estimate of systematic differences between the modalities .
the phantom study was performed to assess the accuracy of ct and spect co - registration .
the jaszczak phantom cylinder interior dimension of 8.5 diameter 7.32 heights ( 21.6 cm 18.6 cm ) containing variable dimension cold rods and solid spheres were used in this study .
the phantom was filled with water and labeled with technetium 99 m ( tc ) of 15 mci .
the spect study was carried out in the gamma camera ( sofa vision medical ( smv ) dstxl , ge health care ) with low - energy high resolution collimators .
the spect study was performed with 64 projections , matrix size of 128 128 , and a pixel resolution of 4.6 mm .
the radiotherapy planning ct ( picker pq 5000 ct scanner , philips medical system ) of the phantom performed in the same orientation with the slice thickness 5 mm , matrix size of 512 512 pixels , and 430 mm field of view correspond to a pixel resolution of 0.84 mm .
the spect matrix was re - sampled to 512 512 matrixes as per the ct data and then co - registered using the statistical parametric mapping software ( spm 2 ) developed by department of neurosciences , university of london software in the matlab platform .
the co - registration accuracy between the spect and the ct data was < 2 mm .
radiotherapy planning ct scans were acquired using three ct / spect compatible markers ( multi - modality markers ) on the patient 's chest in the treatment position using a flat board .
lung perfusion spect images were acquired in a separate session with the same markers at the same position tattooed during ct images .
lung perfusion scans were acquired after an intravenous injection of 200 mbq of tc labeled macro - aggregated albumin using gamma camera on a flat couch , in free breathing .
thereafter both ct and spect images were co - registered using spm 2 software ( statistical parametric mapping ) under matlab 7 platform .
the registration method used here is based on rigid - body model work by collignon et al .
a rigid - body 3d transformation can be parameterized by three scalar translations and rotations matrix perpendicular to each other .
the ct data were kept as a reference and the transformed spect image was processed and re - sliced to a series of registered images so that they match the first image selected voxel - for - voxel .
the accuracy of the co - registration was assessed by superposition of the markers in both the images .
the co - registered ct data with spect was opened using mricro software and the region of interest ( roi ) were delineated ( areas of perfused lung ) .
this produced a multi - colored image , which allowed more accurate volume contouring around a chosen color .
the threshold level was adjusted individually for each patient to match the size of the spect image to the lung volumes defined on ct .
the rois were overlaid on the ct and the data set was transferred on to the treatment planning system ( isis 3d , technology diffusion , france ) for designing the beam portals .
a phantom study was performed to validate the accuracy of the registration algorithm [ figures 1 and 2a d ] .
the accuracy of the registration was evaluated by the superposition of the fiducial markers of the single photon emission computed tomography overlaid image on the computed tomography the accuracy of the registration was evaluated using a phantom .
( a ) jaszezak phantom , ( b ) computed tomography ( ct ) image , ( c ) single photon emission computed tomography ( spect ) image , and ( d ) fused ct spect image
for treatment planning purposes , the following areas were outlined for each patient on the co - registered images in the planning system ; gross tumor volume , body outline , anatomic whole lung ( wl ) lung volume based on ct images as a single organ excluding gross target volume ( gtv ) , functional whole lung based on lung volumes visible on spect images ( fl ) , right lung and left lung , and normal structures . the clinical target volume was created using a 2 cm uniform margin around the gtv . planning target volume ( ptv ) was created with an additional 1.5 cm margin for beam characteristics , setup uncertainties , and organ motion .
fl outlines drawn using the spect images were validated by a nuclear medicine expert in functional imaging .
conventional treatment plans were designed to deliver 60 gy to the ptv with an intent to minimize the dose to the ct defined whole lung ( anatomical wl ) or spect defined whole lung ( fl ) to a dose less than 20 gy or 30 gy .
two treatment plans were created for each patient , one an anatomic plan based on ct scan data alone and the other a functional plan with the incorporation of fl information [ figure 3 a
plan comparison was done using dose volume parameters , v20 ( volume of lung receiving 20 gy ) , v30 ( volume of lung receiving 30 gy ) , and mean lung dose ( mld ) .
for each patient the following data were computed : gtv , whole lung volume ( wlv ) , functional lung volume ( flv ) , ptv95 ( % volume of ptv covered by the 95% isodose ) .
dose volume parameters recorded due to ct - based plan were wl_v20 , wl_v30 , and mean lung dose ( wlmld ) . due to spect - based plan
the following data were computed : fl_v20 , fl_v30 , and mean lung dose functional lung mean lung dose ( flmld ) .
comparison of the dose distribution of ( a ) anatomic and ( b ) functional plan along with the respective dose volume histograms ( c and d )
the difference between dose volume parameters for the anatomic and functional plans was compared using bland - altman plots . in bland - altman plots ,
the differences between the methods that were compared were plotted against their means and gave an unbiased estimate of systematic differences between the modalities .
the phantom study validated the qualitative accuracy of the registration algorithm [ figures 1 and 2a d ] .
the mean gtv volume was 282.76 cm ( range : 82.4 - 571 cm ) .
all patients had a smaller flv when compared with the wlv [ table 1 ] .
the mean difference between wl and fl was 824.7 cm ( range : 1 - 1,618 cm ) [ table 2 ] .
most of the perfusion defects were in the region of tumor and or adjacent to the tumor .
perfusion was either non - uniform with considerable inhomogeneity of fl often due to pre - existing chronic lung dysfunction ( due to old tuberculosis ) or demonstrating specific defects usually due to local atelectasis and copd .
dose volume data ( v20 , v30 , mld ) for both anatomic and functional plans are given in table 1 .
it is intuitive that a large gtv and large ptv would yield higher v20 , v30 and mld as seen in all anatomic plans .
tumors in upper lobe or periphery had lower dose volume parameters than those at hilum , lower or middle lobe .
due to functional plan , dose volume parameters decreased in all patients except in patient 1 and 9 [ table 1 ] .
the mean difference in all the dose volume parameters is depicted in the bland - altman plots [ figure 4 ] [ table 2 ] ( mean change in wl_v20:7.94 gy , fl_v20:6.05 gy , wl_v30:7.05 gy , fl_v30 : 7.55 gy , wlmld : 7.36 gy , flmld : 7.94 gy [ table 2 ] .
bland - altman plots representing the mean value of various dose volume parameters plotted on x - axis and the difference in parameter between anatomic and functional plan on y - axis comparison of anatomical and functional plan comparison of anatomical and functional plan : mean difference in all parameters the mean follow - up of all patients was 9 months .
only four of the evaluable patients were alive at the time of reporting . among these four patients ,
three were alive with progression of disease at 16 , 6 , and 10 months follow - up , and one patient was disease - free at a follow - up of 10 months .
a follow - up spect scan in those with disease progression did not reveal any change in perfusion , whereas in the patient with disease - free status reperfusion in area adjacent to the region of target was observed .
the main aim of this study was to assess whether the incorporation of fl information into conventional radiotherapy planning could result in reduction of dose to healthy functioning lung .
the validity of the dose - volume data depends on the method of co - registering spect lung perfusion images with ct images .
the phantom study showed reasonable qualitative matching of fiducial markers of spect and ct images .
co - registration of images using public domain software has its utility in developing country like ours where procurement of co - registration software involves dear investments .
as previously shown and confirmed in this study , not all regions of the anatomic lung defined by ct scans are of equal physiologic importance .
most of the patients had perfusion defects in the region of the tumor ( 54% ) or in and adjacent to tumor region ( 36% ) , a non - functioning lung in the entire ipsilateral hemithorax in the rest .
since most of the perfusion defects are in the tumor - bearing lung , in principle one should attempt to direct all beams through the ipsilateral lung to avoid unnecessary irradiation of opposite lung .
this principle can be brought to an advantage even in centers which do not have the facility of fl imaging as in most of the radiotherapy centers in our country .
the incorporation of fl information into the conventional treatment plans aided in diversion of the beams away from the fl .
seppenwoodle demonstrated the feasibility of perfusion - weighted optimization of treatment plans and suggested improvement in mean lung doses by an increase in the weights of those beams that were directed through hypoperfused lung regions .
he concluded that perfusion - weighted optimization should result in clinical benefit in patients with large perfusion defects and larger target volume .
spect perfusion information has also been incorporated in inverse radiotherapy planning for lung cancer and it was found that spect was warranted only in those patients with large perfusion defects .
mcguire reported a methodology for using spect to deliver intensity - modulated radiation therapy ( imrt ) , by segmenting healthy lung into four regions on the basis of spect intensity and they found a reduction in v20 and v30 values of 15.5% and 10.5% respectively .
the use of imrt when compared with 3-dcrt improved the avoidance of fl defined by perfusion spect scan in selected patients and imrt allowed for effective dose escalation by specific avoidance of fl .
incorporation of functional information seems to be beneficial as far as planning studies are concerned .
but its utility in the clinic needs to be defined according to different presenting stage .
almost all the patients attending our clinic were stage iii or iv patients with target volumes almost three to eight times than those reported by other authors .
many of these patients could have been upstaged to stage iv disease with the availability of pet , which explains the shorter survivals ( 9 months mean follow - up ) in our setup when compared with other studies where reported median survivals were 15 - 18 months . by the time we expect radiation pneumonitis to manifest ( 3 - 6 months or more )
our patients have either succumbed to disease or have had progression of disease or recurred .
hence the clinical benefit of perfusion spect in inoperable locally advanced lung cancer could not be ascertained .
it should be beneficial in patients with smaller target volumes with large perfusion defects . perhaps intensity - modulated radiotherapy in appropriately staged iii patients with a constraint on fl
spect perfusion images can be accurately co - registered with radiotherapy planning ct scans using public domain software .
information regarding fl does aid in diverting beams away from the fl which might minimize radiation pneumonitis . | purpose : lung perfusion scan provides a map of the spatial distribution of lung perfusion . this can be used to design radiation portals to spare functional lung ( fl ) , potentially reducing lung toxicity .
the purpose of this study was to assess the utility of lung perfusion single photon emission computed tomography ( spect ) in treatment planning for lung cancer patients.materials and methods : radiotherapy treatment planning computed tomography ( ct ) scans and spect scans of 11 patients of lung cancer suitable for external radiotherapy were co - registered .
conventional treatment plans ( anatomic plan ) and plans with fl information ( functional plan ) was generated .
the difference in dose volume parameters ( v20 , v30 and mean lung doses ) due to these two plans were compared using bland - altman plots.results:functional plans produced a more favorable plan compared with anatomic plan in all except three cases .
fl v20 values and fl mean lung dose were reduced for all patients by an average of 5.45 gy and 7.72 gy respectively which were statistically significant.conclusions:lung perfusion scans provide functional information which is not provided by ct scans .
spect - guidance aids in reducing the dose delivered to highly perfused regions which could reduce the incidence of pneumonitis . |
polymicrogyria is a malformation of cortical development in which the process of normal cerebral cortical development is disturbed late in the stage of neuronal migration or early in the stage of cortical organization ; thus , it is considered a disorder of neuronal organization . as a result of these disturbances to the developmental process ,
the deeper layers of the cerebral cortex develop abnormally and multiple small gyri form within the cortex .
polymicrogyria has a range of histologic appearances , all having in common a derangement of the normal six - layered lamination of the cortex , an associated derangement of sulcation , and fusion of the molecular layer across sulci [ 35 ] . in areas of polymicrogyria ,
causes of polymicrogyria include congenital infection ( particularly cytomegalovirus infection [ 6 , 7 ] ) , localized or diffuse in utero ischemia [ 8 , 9 ] , or mutations ( table 1 ) [ 1023 ] .
patients with polymicrogyria may have a wide variety of clinical presentations , ranging from hemiparesis or partial epilepsy to developmental delay , quadriparesis , and medically refractory , intractable epilepsy ; the neurologic disability appears to depend upon the portion[s ] of brain involved , but may also depend upon the type of polymicrogyria and the presence or absence of associated anomalies .
neurologic manifestations are similar among patients who have polymicrogyria due to different causes [ 6 , 22 , 24 , 25 ] .
the severity of the clinical presentation and age at presentation depend mostly upon the extent of cortical involvement ; bilateral involvement and involvement of more than half of a single hemisphere are poor prognostic indicators , portending moderate to severe developmental delay and significant motor dysfunction .
polymicrogyria affects variable portions of the cerebral cortex : it may be focal , multifocal , or diffuse ; it may be unilateral , bilateral , and asymmetrical ; or bilateral and symmetrical .
the most common location ( in 6070% of cases ) is around the sylvian fissure , particularly the posterior aspect of the fissure ; however , any part of the cerebral cortex , including the frontal , occipital , and temporal lobes , can be affected [ 3 , 2428 ] .
polymicrogyria may be an isolated malformation or it may be associated with other brain malformations ; when other malformations are identified , the most common are corpus callosum agenesis and hypogenesis , cerebellar hypoplasia , periventricular nodular heterotopia , and subcortical heterotopia .
some of the best known of these are aicardi syndrome ( omim 304050 [ 32 , 33 ] ) , delleman syndrome ( oculo - cerebral - cutaneous syndrome , omim 164180 [ 34 , 35 ] ) , digeorge syndrome ( omim 188400 , also called the 22q11.2 deletion syndrome in which several genes are deleted ) , warburg micro syndrome ( omim 600118 [ 3638 ] ) , and d - bifunctional protein deficiency ( omim 261515 ) .
table 2multiple congenital anomaly syndromes with polymicrogyria.syndromeanomaliesgeneaicardi syndromecallosal agenesisnot knownpolymicrogyriaheterotopia [ periventricular and subcortical]retinal anomaliesdelleman syndromecallosal agenesisnot knowncystic microphthalmiafrontal polymicrogyriaperiventricular heterotopialarge , dysplastic tectumabsent cb vermisdigeorge syndromeparathyroid hypoplasia22q11.2thymic hypoplasiacleft palatecardiac malformationsfacial anomaliespolymicrogyria [ perisylvian , right hemisphere preferred]warburg micro syndromemicrocephaly2q21.3microcorneacongenital cataractoptic atrophyhypogenitalism with hypotoniacallosal hypo- or agenesisfrontoparietal polymicrogyriad - bifunctional protein deficiencygeneralized osteopenia5q2bilateral perisylvian polymicrogyrialarge anterior fontanellefrontal bossingup - slanting palpebral fissureshypertelorism multiple congenital anomaly syndromes with polymicrogyria .
this variability is most likely a result of three factors : imaging factors ( amount of gray matter
white matter contrast , thickness of the slices ) ; the stage of maturity / myelination of the brain at the time of the imaging study ; and , in all likelihood , the type of pmg . in a previous analysis of pmg , i noted that it can have a coarse appearance or a delicate appearance and that the appearance seen in the so - called cobblestone malformations differs from that seen in most bilateral polymicrogyria syndromes .
the precise reasons for these differing appearances are not known , but the development of the cerebral cortex is so complex that it is not surprising that disruptions of the processes of late cortical migration and cortical organization at different stages might result in slightly different malformations .
sometimes the cortex appears thick and coarse ( with an appearance of palisades of cortex , ( fig . 4 ) ) , while other times the microgyri appear fine and delicate ( fig . 1 )
, even when the brains are similarly myelinated . however , these variations in appearance may not be detectable on routine , 5-mm thick images .
therefore , images with thin sections and optimal gray matter white matter contrast [ we acquire volume 3dft spoiled gradient acquisition ( t1 weighted ) and volume 3dft fast spin echo ( t2 weighted ) images , both in the sagittal plane with 1.5 mm partition size ] should always be acquired .
evaluation in three planes is often necessary to detect irregularities of the gray matter white matter junction , which are often the most convincing evidence of dysplastic brain ( fig .
3 ) ; this is most easily accomplished via volumetric acquisition with display in all three orthogonal planes ( sagittal , axial , and coronal ) .
the volume acquisitions can be displayed as three - dimensional surface images ( fig .
5 ) and can be utilized for stereotactic localization , aiding in surgical therapy , if appropriate .
the inner surface of the polymicrogyric cortex looks thin [ 23 mm ] and bumpy , while in myelinated areas it looks thicker ( 58 mm ) and relatively smooth .
the reason proposed for this is that a 45 mm layer of gliotic white matter runs through the polymicrogyric cortex , blending in with white matter in the unmyelinated brain and blending in with cortex after myelination . finally , as stated earlier , polymicrogyria is almost certainly a heterogeneous malformation that can have many different appearances : thick and coarse , fine and delicate , with shallow or deep sulci .
whatever the reason , it is important to realize that a spectrum of cortical appearances , all having some sort of small gyri , can be seen in patients with polymicrogyria .
fig . 1parasagittal ( a ) and axial ( b ) t1-weighted images show the delicate appearance of pmg ( black arrows ) in the right sylvian and suprasylvian cortex .
note the continuity of the posterior sylvian fissure on the parasagittal image ( a ) ; this is diagnostic of perisylvian pmgfig .
parasagittal ( a ) and axial ( b ) images show coarse pmg in the parieto - occipital ( small black arrows ) and perisylvian ( large black arrows ) regionsfig .
3axial fse t2 weighted image shows right frontal pmg ( white arrows ) with fusion of the molecular layer of cortex resulting in paradoxically smooth cortical surfacefig .
4axial t1-weighted image shows diffuse coarse pmg with an appearance of palisades of cortexfig .
the surface rendering ( b ) shows the absence of normal sulci and the bumpy cortical surface in the affected area ( arrows ) parasagittal ( a ) and axial ( b ) t1-weighted images show the delicate appearance of pmg ( black arrows ) in the right sylvian and suprasylvian cortex .
note the continuity of the posterior sylvian fissure on the parasagittal image ( a ) ; this is diagnostic of perisylvian pmg thick and irregularly bumpy , coarse appearance of pmg .
parasagittal ( a ) and axial ( b ) images show coarse pmg in the parieto - occipital ( small black arrows ) and perisylvian ( large black arrows ) regions axial
fse t2 weighted image shows right frontal pmg ( white arrows ) with fusion of the molecular layer of cortex resulting in paradoxically smooth cortical surface axial t1-weighted image shows diffuse coarse pmg with an appearance of palisades of cortex surface rendering of pmg from volumetric acquisition of data .
a image shows a parasagittal image of extensive perisylvian coarse pmg ( arrows ) . the surface rendering ( b ) shows the absence of normal sulci and the bumpy cortical surface in the affected area ( arrows ) in addition to the variability of the appearance of polymicrogyria ,
. it may be superficial , with the cortex appearing flat and congruent to the arc of normal cortex , or may course radially inward , as if it were buckled or folded toward the ventricle .
this infolding of cortex may be small or large , but close examination will show that the cortical features are similar [ bumpy , irregular inner and outer cortical surfaces ] in both the superficial and radially folded types .
the cortex surrounding the sylvian fissures is involved in 80% of cases , with the frontal lobe being most commonly involved ( 70% ) , followed by parietal ( 63% ) , temporal ( 38% ) , and occipital ( 7% ) lobes .
the striate cortex , cingulate gyrus , hippocampus , and gyrus rectus are typically spared .
polymicrogyria is sometimes calcified , but it is difficult to know the frequency because few patients are studied with ct . it is not clear if the calcification is of any significance . finally , it is important to recognize that anomalous venous drainage is common in areas of dysplastic cortex , seen in up to 51% of patients with polymicrogyria .
large vessels are especially common in regions where there is a large infolding of thickened cortex
. such large vessels , when seen in association with abnormal , thickened cortex , should not be mistaken for vascular malformations .
several syndromes of bilateral symmetrical polymicrogyria have been described ; these should be recognized by neuroimagers .
the best known and most common of these is bilateral perisylvian polymicrogyria ( also called congenital bilateral perisylvian syndrome ) .
the heterogeneous inheritance patterns suggest that mutations of several different genes can cause this malformation ; three locations ( xq21.33-q23 ( srpx2 ) , 22q11.2 , and xq28 ) have been identified [ 13 , 19 , 20 , 22 ] .
a syndrome of developmental pseudobulbar palsy ( oropharyngeal dysfunction and dysarthria , 100% ) , epilepsy ( 8090% ) , mental retardation ( 5080% ) , and , sometimes , congenital arthrogryposis has been described [ 44 , 4648 ] .
other patients are brought to attention in infancy or early childhood because of delayed development ( 60% ) , palatal dysfunction ( 40% ) , hypotonia ( 30% ) , arthrogryposis ( 30% ) , or motor deficits ( 25% ) [ 47 , 49 ] .
seizures are present in 4060% and may be of many clinical types [ 47 , 49 ]
. studies of familial cases of congenital bilateral perisylvian polymicrogyria show a lower incidence of these clinical manifestations [ 45 , 50 ] , possibly because patients with minimal symptoms are more readily identified and examined
. a developmental reading disorder and reading impairment without severe motor or cognitive handicap may be the cause of seeking medical attention in this group .
bilateral perisylvian polymicrogyria can be graded according to severity on mri ( with grade 1 the most severe and grade 4 the mildest ) : grade 1 , with perisylvian polymicrogyria extending to the frontal or occipital pole ( fig .
6 ) ; grade 2 , with polymicrogyria extending beyond the perisylvian region but not to either pole ( most common , fig . 7 )
; grade 3 , with polymicrogyria of the perisylvian region only ( fig . 8) ; and grade 4 , with polymicrogyria restricted to the posterior perisylvian regions .
parasagittal ( a ) and axial ( b ) t1-weighted images show coarse polymicrogyria extending from the frontal pole to the occipital polefig .
parasagittal ( a ) and axial ( b ) images show coarse perisylvian polymicrogyria sparing the anterior frontal lobes and the occipital lobesfig .
parasagittal t1-weighted images ( a ) and axial t2-weighted images ( b ) show polymicrogyria limited to the insulae and operculae grade 1 perisylvian pmg .
parasagittal ( a ) and axial ( b ) t1-weighted images show coarse polymicrogyria extending from the frontal pole to the occipital pole grade 2 perisylvian pmg .
parasagittal ( a ) and axial ( b ) images show coarse perisylvian polymicrogyria sparing the anterior frontal lobes and the occipital lobes grade 3 perisylvian pmg .
parasagittal t1-weighted images ( a ) and axial t2-weighted images ( b ) show polymicrogyria limited to the insulae and operculae bilateral frontoparietal polymicrogyria is another well - defined syndrome [ 12 , 52 ] .
affected patients are characterized by global developmental delay of at least moderate severity , seizures , disconjugate gaze , and bilateral pyramidal and cerebellar signs .
mr demonstrates symmetric cortical dysgenesis affecting the frontoparietal regions most severely , as well as ventriculomegaly , bilateral white matter signal changes , and small brainstem and cerebellar structures with dysmorphic cerebellar cortex ( fig .
the presence of the white matter changes and the posterior fossa anomalies is very uncommon for polymicrogyria syndromes .
in addition , the cortical malformation itself has a slightly different appearance than that of most polymicrogyria .
indeed , the overall appearance of the brain is more similar to that of the so - called cobblestone malformations , associated with congenital muscular dystrophies and resulting from abnormalities of linkage of radial glial cells to the pial basement membrane overlying the developing cortex [ 53 , 54 ] .
further research has shown that the pial basement membrane is regulated by gpr56 , the gene that causes bfpp when mutated , and that mutations of gpr56 cause gaps in the pial basement membrane and abnormal linkage of radial glial cells to that membrane .
moreover , this observation raises the question of what precisely defines polymicrogyria and whether the different types of polymicrogyria should be analyzed and distinguished .
axial ( a ) and coronal ( b ) t2-weighted images show a different appearance to the cortex , that of multiple radially oriented neuronal components separated by fibro - glial stroma .
note the prominent cerebellar fissures and the subcortical cerebellar cysts ( arrows ) bilateral frontoparietal polymicrogyria .
axial ( a ) and coronal ( b ) t2-weighted images show a different appearance to the cortex , that of multiple radially oriented neuronal components separated by fibro - glial stroma .
note the prominent cerebellar fissures and the subcortical cerebellar cysts ( arrows ) other syndromes of bilateral symmetrical polymicrogyria have been described .
several groups have described patients with spastic quadriparesis and epilepsy , on whom imaging shows bilateral symmetrical frontal polymicrogyria ( fig .
guerrini et al . have described patients with bilateral medial parietal - occipital polymicrogyria ( fig .
2 ) ; thus , it appears that any region of cortex may be involved by bilateral , symmetrical polymicrogyria .
a syndrome of congenital hemiplegia and epilepsy has been described in patients with large areas of unilateral polymicrogyria .
parasagittal ( a ) and axial ( b ) t1-weighted images show coarse parasagittal infoldings ( arrows ) of polymicrogyria bilateral frontal polymicrogyria .
parasagittal ( a ) and axial ( b ) t1-weighted images show coarse parasagittal infoldings ( arrows ) of polymicrogyria a syndrome of megalencephaly with polymicrogyria and hydrocephalus has been described .
affected children present at birth or in early infancy with macrocephaly and hypotonia ; delayed motor and cognitive development become evident as they grow .
epilepsy , postaxial polydactyly , cutis marmorata , midface capillary malformation , and coarse facial features are frequently present [ 6165 ] .
imaging reveals ventriculomegaly ( from hydrocephalus ) and polymicrogyria , which is most severe in the perisylvian region ( typically grade 1 or 2 ) .
this disorder is now thought to include those previously called megalencephaly polymicrogyria polydactyly hydrocephalus syndrome and macrocephaly capillary malformation syndromes [ 6365 ] . in summary ,
polymicrogyria is not a single entity , but a group of disorders that has many causes and many clinical and radiological phenotypes .
imaging characteristics may be useful in helping to differentiate the different types of polymicrogyria , which may appear as one component of multiple congenital anomaly syndromes or as isolated cns malformation .
it is important for neuroimagers to acquire high - quality images in affected patients and carefully analyze the images to help to optimize the diagnosis and treatment . | polymicrogyria is one of the most common malformations of cortical development .
it has been known for many years and its clinical and mri manifestations are well described .
recent advances in imaging , however , have revealed that polymicrogyria has many different appearances on mr imaging , suggesting that is may be a more heterogeneous malformation than previously suspected .
the clinical and imaging heterogeneity of polymicrogyria is explored in this review . |
according to lekholm and zarb,1 the bone density of the posterior maxilla is classified as type 4 bone , which means that a thin layer of cortical bone surrounds low - density trabecular bone .
implants placed in soft bone or d4 bone require a longer healing period for better bone remodeling and maturation .
a poor bone density does not provide support for the implant during the healing and loading stages.24 higher implant failure rates have consistently been found in areas of poor bone quality , such as the posterior segment of the maxilla.5,6 there are several special considerations that should be taken into account when implants are placed in the posterior maxillary area , including drilling an undersized preparation to obtain primary stability , using a modified ( roughened ) implant surface , using a wide diameter and aggressively designed implant , and using a submerged technique to avoid implant loading stress .
all of these measures result in an increased implant surface area that enhances bone implant contact and implant stability.3,7,8 the progressive implant loading technique was developed by misch9 for implants placed in areas with poor bone density .
this technique allows bone to mature during the loading period without overloading the implant and resulting in bone loss or implant failure.10 ban et al11 conducted a study comparing the differences between progressive , immediate , and delayed loading of implants .
the result was a significant increase in bone implant contact and a decrease in vertical bone loss in the progressive loading group .
maxillary sinus floor perforation during implant placement resulting from overestimation of the bone height has many consequences depending on the severity of injury .
primarily , sinus involvement during implant placement may prevent osseointegration of the apical part of the fixture.12 additionally , soft tissue from the floor of the sinus may grow on the apex of the implant instead of bone , affecting the final prognosis.12 sinus infection and sinusitis may also occur if bacteria proliferate on the apex of the implant surface , affecting the process of bone remodeling.1315 it is also possible for no adverse consequences to occur and for new bone to form around and above the apex of the implant.12 our case report consists of two sections : the first describes maxillary sinus perforations and their effect on implant prognosis and survival , and the second discusses the novel technique of immediate progressive implant loading .
the patient was a 52-year - old woman with no history of systemic disease or parafunctional occlusion .
the patient presented to our facility with a main request to replace her missing maxillary left second premolar and first molar with implants .
an orthopantomogram was taken ( figure 1 ) , and after clinical and radiographic examination , a taper implant with a 4.0 mm width and a 10 mm length was selected for replacement of both teeth . using local anesthesia ,
the bone was then exposed and prepared according to the manufacturer s recommendations . during preparation of the first molar socket ,
the resistance to drilling decreased , indicating sinus floor penetration . because the bone density was poor , the final drill was inserted halfway , and two implants with a diameter of 4.0 mm and length of 10 mm were placed , replacing the first molar and second premolar .
the implant design was a tapered , modified surface ( sand - blasted , large grit , acid - etched [ sla ] ) that was threaded to the top ( superline , dentium , cypress , ca , usa ) to achieve better implant stability and increase the implant surface area in contact with the surrounding bone .
implants were placed approximately 1 mm subcrestally , and resonance frequency analysis ( rfa ) measurements were taken for both implants ( table 1 ) .
the reading for the second premolar implant was 73 isq ( implant stability quotient ) in all four directions , and for the first molar implant , the reading was 69 isq in all directions except for the buccolingual direction , which was 64 isq .
a cover screw was placed on the second premolar implant and covered with gingival tissue to obtain primary closure and allow healing using the submerged technique .
the first molar implant was immediately loaded with a temporary plastic abutment , and a crown was fabricated from a light - cured composite resin material ( figure 2 ) . at this stage ,
the crown was out of occlusion , with a narrow occlusal table and no interproximal contact ( figures 3 and 4 ) .
the tissue flap was adapted to the temporary crown , and the gingiva was sutured around it ( figure 5 ) .
a periapical radiograph was taken postoperatively for both implants and was considered a basic radiograph ( figure 6 ) . the radiograph shows the implant in the molar area penetrating the maxillary sinus approximately 23 mm .
postoperative instructions were given , and after 10 days the patient returned for suture removal and follow - up .
no signs or symptoms of maxillary sinus infection or inflammation were reported by the patient .
the rfa measurements taken at that time were 73 isq in the mesiodistal and distomesial directions and 70 isq in the buccolingual and lingobuccal directions .
the crown was modified ( figure 7 ) by increasing the width of the occlusal table and obtaining distal contact with the adjacent tooth ( upper right second molar ) . at this stage ,
again , the patient was examined for any signs or symptoms of maxillary sinus infection , and none were found .
two months after implant placement , the patient returned for the third stage of crown modification .
the temporary abutment and crown were removed , and the rfa measurements taken at this time were 75 isq in the mesiodistal and distomesial directions and 70 isq in the buccolingual and lingobuccal directions .
the crown was modified by increasing the vertical height and obtaining occlusal contact with the opposing tooth in the axial direction only ( figure 8) .
occlusal contacts during excursive movements were removed , and only central contact remained . during the same appointment , the second premolar implant was uncovered , and the rfa measurements taken were 76 isq in the mesiodistal and distomesial directions and 66 isq in the buccolingual and lingobuccal directions . a healing abutment was placed and left for 1 month to allow healing of the soft tissue ( figure 9 ) .
three months after implant placement ( figure 10 ) , the rfa measurement for the first molar was 75 isq in the mesiodistal , lingobuccal , and distomesial directions and 71 isq in the buccolingual direction . for the second premolar , the rfa values were 75 isq in the mesiodistal direction , 79 isq in the distomesial direction , and 70 isq in the buccolingual and lingobuccal directions .
impressions were taken at this appointment using a closed - tray technique and hex coping .
jaw relationships and bite registration were obtained using a wax bite rim and o - bite .
titanium hex abutments were placed using 30 ncm of force for both implants , and provisional crowns were cemented with temporary cement ( figure 11 ) .
occlusion was checked and adjusted , leaving only centric occlusal contacts and removing all contacts found during excursive movements ( figure 11 ) .
maxillary sinus involvement during implant placement may lead to decreased contact of the implant with bone and prevent osseointegration of the apical portion of the fixture.12 soft tissue from the floor of the sinus may grow instead of bone , which affects the prognosis.12 additionally , sinus infection and sinusitis may occur1315 due to bacterial growth on the apex of the implant surface , making bone formation and osseointegration impossible .
some authors recommend engaging the apex of the implant with the sinus floor to obtain increased implant stability because the sinus floor is composed of dense cortical bone.16,17 zhong et al12 conducted histological studies on canines , concluding that penetration of dental implants into the maxillary sinus with membrane perforation does not necessarily compromise implant osseointegration and sinus health during the 5-month observation period.12 the same researchers concluded that if the implant apex protruded 2 mm or less into the sinus , it was possible for healthy maxillary bone to regenerate over the apex of the implant .
however , when the perforation was more than 3 mm , the soft tissue formed a cuff around the implant.12 this effect may be due to elasticity of the schneiderian membrane , which can stretch approximately 2 mm before perforating .
the second part of this case report describes a progressive implant loading technique that has been modified by the author .
progressive implant loading was developed by misch9 and is recommended for implants that have been placed in soft bone or grafted sites . after the osseointegration period , the implant is exposed and progressively loaded to prevent overloading while simultaneously increasing bone density .
this case report illustrates how progressive loading with staging increases the width of the occlusal table and how the height of the crown can increase implant stability , especially if performed within the first 4 weeks .
many studies have shown that there is decreased implant stability during the first 4 weeks of the healing period due to active bone remodeling and new immature bone formation.1820 when we compared immediate progressive loading of implants with low primary stability to submerged healing of implants with higher primary stability , rfa showed an increased reading of 72 isq for the loaded implant , while a decrease in implant stability was found for the submerged implant with a reading of 71 isq after a 2-month healing period .
this provides additional evidence that progressive loading of implants can stimulate bone formation and increase bone density compared with placement of implants using the conventional technique of delayed loading .
ban et al13 reported that progressive implant loading can accelerate the mineralization process during the first 28 days of the healing period . placing an implant into soft bone and immediately loading it
is considered a risky procedure10 because soft bone does not support implants well ; consequently , protective measures should be taken.2,3,13,19,20 protective measures for achieving primary stability10 include choosing a wider diameter implant to increase the surface area and bone implant contact.6,11 aggressive implant designs , which entail deep threads that extend to the top , allow the implant to engage additional bone during placement due to an increased surface area.21 special considerations for socket preparation include inserting the last drill halfway or occasionally skipping the last drill so that the implant is placed in an undersized socket.6,10 tapered implant designs allow the coronal part of the implant to engage the cortical bone of the alveolar crest .
placing implants subcrestally by approximately 1 mm provides additional protection to the implant and allows increased engagement of the cortical bone.22,23 another factor related to implant stability is the surface of a treated implant .
many studies have shown that implants with an sla surface can enhance bone formation and act as a chemotactic factor for osteogenic cells.24 sla surfaces may enhance the quality of soft bone surrounding the implant by increasing its density.25,26 all of the above factors should be considered when implants are placed in soft bone and immediately loaded .
the concept for this technique originated from the process of tooth eruption : during eruption , the tooth has a partially developed root , and when it finally erupts into occlusion with its antagonist , only three quarters of the root has been formed.26 similarly , with this technique , we attempt to make the implant erupt into the oral cavity by progressively increasing the crown width and height . this process allows time for the bone to grow and mature with the increased loading stimuli , resulting in implant stability and increased bone density . in other words ,
progressive implant loading can be used as a safe technique for the immediate loading of implants with maxillary sinus involvement .
no adverse consequences were found when the maxillary sinus floor was perforated , provided that the membrane was intact and healthy and the implant protrusion into the sinus was within 2 mm . | the displacement of a dental implant into the maxillary sinus may lead to implant failure due to exposure of the apical third or the tip of the implant beyond the bone , resulting in soft tissue growth .
this case report discusses dental implant placement in the upper first molar area with maxillary sinus involvement of approximately 2 mm .
a new technique for progressive implant loading was used , involving immediately loaded implants with maxillary sinus perforation and low primary stability .
follow - up was performed with resonance frequency analysis and compared with an implant placed adjacent in the upper second premolar area using a conventional delayed loading protocol .
implants with maxillary sinus involvement showed increasing stability during the healing period .
we found that progressive implant loading may be a safe technique for the placement of immediately loaded implants with maxillary sinus involvement . |
weight loss among elderly patients is a common clinical problem . for patients with more than 4% weight loss ,
2-year survival rate is less than 72% , and 3-year survival rate is less than 65% ( wallace et al 1995 ; wallace and schwartz 1997 ) .
this manuscript will summarize the results of our studies and discuss and review the general usage of megestrol acetate ( ma ) in the treatment of weight loss in cachectic elderly , cancer , and hiv patients .
roberts and colleagues ( roberts et al 1994 , 1997 ; roberts 1995 , 2000 ) investigated the effects of aging on the mechanism of body energy regulation and thereby determined the causes of unexplained weight loss in older persons .
they found that aging may be associated with a significant impairment in the ability to control food intake following overeating or undereating .
hospitalization resulting in undereating can have a devastating effect on the frail elderly ( sullivan et al 1999 ) .
studies in both cancer patients and hiv patients have shown that ma can improve appetite and help patients gain weight ( tchekmedyian et al 1987 , 1991 ; oster et al 1994 ; von roenn 1994 ) .
ma is a synthetic derivative of a naturally occurring progestational agent , which is similar to progesterone .
ma is well tolerated in patients with advanced malignant diseases and has a positive dose - response effect on appetite stimulation ( tchekmedyian et al 1987 , 1991 ; oster et al 1994 ) .
loprinzi et al ( 1994 ) found the optimal dose in their study to be 800 mg / day .
loprinzi et al ( 1993 ) found that this was primarily non - fluid weight .
ma has also been shown to improve body weight , sense of appetite , and performance status in patients with hormone - insensitive cancers ( feliu and gonzalez - baron 1992 ) .
the fda approved in 1994 the use of ma for the treatment of anorexia , cachexia , and/or an unexplained , significant weight loss in patients with a diagnosis of acquired immunodeficiency syndrome ( aids ) .
ma was first produced in a tablet form , then in a concentrated oral suspension form , and most recently , in an oral suspension form developed using nanocrystal technology .
nanocrystal technology was designed specifically to optimize drug delivery and enhance the bioavailability of drugs that have poor solubility in water .
the precise mechanism by which ma promotes weight gain is unclear . proposed mechanisms and their potential side - effects are summarized in figure 1 .
reported results show that ma may act both an progestational agent as well as an anti - inflammatory / glucocorticoid agent ( bojar et al 1979 ; kontula et al 1983 ; loprinzi et al 1992a , 1992b ; pridjian et al 1987 ; selman et al 1996 , 1997 ; chang 1998 ; gomez et al 1998a , 1998b , 2002 ; meacham et al 2003 ) .
lapp et al ( 1995 ) have found , for example , that higher endogenous progesterone concentrations are common in late pregnancy , and those elevated concentrations result in decreased il-6 levels , which are 40%50% of controls .
it makes sense that progesterone plays an important role in improving nutritional status and decreasing localized inflammation by down - regulation of il-6 production during pregnancy .
ma also has been found to decrease cytokine production during the treatment of malignancy ( barak et al 1998 ; mantovani 1997 ; mantovani et al 1998a , 1998b ) .
wiedemann et al ( 1998 ) found that ma dosages between 160 and 480 mg activate progesterone receptors and lead to pituitary hormone secretion of a well - defined progesterone receptor ligand in a non - linear u - shape dose dependency .
our experience in randomized studies indicates that across both placebo and active treatment groups , there is a statistically significant negative correlation between the levels of pro - inflammatory cytokines , such as il-6 and tnf , and various nutritional parameters , body mass indices , improvement in qol , and weight gain ( yeh et al 2000a , 2000b , 2001 ) .
these findings seem to support a role for inflammatory cytokines in the idiopathic cachexia syndrome of the elderly .
ma binds to glucocorticoid receptors ( kontula et al 1983 ) in a possible dual agonist antagonist fashion , acting as a weak agonist , but then serving as an antagonist in blocking the binding of more potent endogenous glucocorticoids ( kontula et al 1983 ; mann et al 1997 ) . structural similarities between ma and glucocorticoids have been invoked to explain glucocorticoid - like activity and increased peripheral resistance to insulin after treatment with the drug ( van veelen et al 1984 , 1985 ; loprinzi et al 1992b ; herkert et al 2001 )
. the dual action of ma may be the possible explanation for the unresolved inconsistencies of megestrol acetate s neuroendocrine - progesterone effects and its potential reported side - effects such as hyperglycemia , hypoglycemia , venous thromboembolism , secondary adrenal suppression , and adrenal insufficiency ( selman et al 1996 , 1997 ; koller et al 1999 ; herkert et al 2001 ; oberhoff 2001 ; mckone et al 2002 ; bennett 2003 ; kropsky et al 2003 ; thomas 2004 ) .
reuben and colleagues reported that all patients had cortisol suppression after 9 weeks on ma ( 70% with ma>400mg / day ) ( reuben et al 2005 ) .
the suppression of the adrenal axis was at the level of the hypothalamus/ pituitary ( kontula et al 1983 ) .
effects on cortisol are reversed in 24 weeks once off ma ( meacham et al 2003 ) .
ma also inhibits con - a and pha stimulated t - cell proliferative response ( kontula et al 1983 ) , making patients with long - term usage of ma vulnerable to infection and malignancy .
short - term ( 12 weeks ) use of the drug appears to be safe ( yeh et al 2000b ; pascual lopez et al 2004 ; simmons et al 2004 ; reuben et al 2005 ) .
ma appears to be one of the more potent appetite stimulants of currently used drugs ( loprinzi et al 1999 ; mwamburi et al 2004 ; jatoi et al 2002 , 2004 ; tomiska et al 2003 ) .
jatoi found that eicosapentaenoic acid ( epa ) supplement , either alone or in combination with ma , is no better than ma alone ( jatoi et al 2002 , 2004 ) .
ma provided superior anorexia palliation among advanced cancer patients compared with dronabinol alone ( jatoi et al 2002 ) , and combination therapy did not appear to confer additional benefit ( jatoi et al 2002 ) .
pascual lopez et al ( 2004 ) did relevant database searches for randomized controlled trials of ma to treat anorexia
data were extracted by two independent reviewers , and meta - analyses were performed where possible ; 26 studies were included ( n = 3887 ) . compared with placebo , ma increased appetite in oncology patients ( pascual lopez et al 2004 ) , led to weight gain ( rr=1.88 [ 95% ci 1.432.47 ] ) and improved quality of life ( hrqol ) ( rr=1.52 [ 95% ci 1.002.30 ] ) . in aids
there were no appreciable differences between lower ( < 800 mg / day ) and higher ( > 800 mg / day ) doses of ma ( pascual lopez et al 2004 ) .
they found the main side - effect of ma was lower limb edema ( rr=1.67 [ ci 1.222.28 ] ) ( pascual lopez et al 2004 ) .
berenstein and ortiz ( 2005 ) also found ma improved appetite and weight gain in patients with cancer in their cochrane database system review .
our experience with ma has shown that its use in treating cachexia in the elderly improves quality of life and weight gain ( yeh et al 2000b , 2001 ) .
we found that ma improved appetite ( yeh et al 2000b ) and had a tendency to improve weight gain .
furthermore , it improves quality of life ( yeh et al 2000a , 2000b ) . during the study , reduction of pro - inflammatory cytokine levels
was associated with improved quality of life , as well as increased appetite , lean muscle mass , and overall weight ( yeh et al 2000b , 2001 ) .
increasing prealbumin correlated with reduction in tnfr - p55 and tnfr - p75 ( yeh et al 2000b , 2001 ) .
there was no difference in adverse effects between two groups ( yeh et al 2000b ) . increasing weight / fat free mass correlated with reduction in sil-2r and tnfr - p75 ( yeh et al 2001 ) . improving appetite correlated with quality of life improvement , weight gain , and nutritional indicator changes ( yeh et al 2000a , 2000b , 2001 ) .
reduction in one pro - inflammatory cytokine was highly correlated with reduction in the other cytokine levels ( yeh et al 2001 ) .
the factors that could predict survival among the subjects were higher prealbumin , albumin , and weight gain ( yeh et al 2000b , 2001 , 2004 ) . elevated il-6 , crp , tnfr - p55 and tnfr - p75 were associated with decreased survival ( yeh et al 2000b , 2001 , 2004 ) . however
, no significant differences in survival were noted between the ma - treated and the placebo groups ( p=0.72 ) , as estimated by the kaplan - meier method ( yeh et al 2000a , 2000b , 2001 , 2004 ) . in our experience , most patients had improved appetite by 6 weeks with ma treatment , although the weight gain was not yet significant at that time ( yeh et al 2001 , 2000b ) .
a course of treatment with ma for 12 weeks is probably enough to improve the appetite that will result in eventual weight gain . by 6 months ,
most of the treated patients gained weight , and there was a trend for this weight gain to be in the form of fat . increased fat mass
has been noted in patients with cancer and aids following treatment with ma ( von roenn et al 1988 ; loprinzi et al 1993 ; von roenn 1994 ; von roenn and knopf 1996 ) .
dulloo and colleagues ( dulloo et al 1997 ; dulloo 1998 , 1997 ) found that replenishment of fat stores was the initial event following feeding of volunteers previously subjected to semi starvation and reported that this observation could be generalized to recovery from other forms of refeeding after weight loss .
they attributed the preferential gain in fat to reduced thermogenesis and changes in energy partitioning during recovery from starvation .
the initial gain in fat mass was related to the pre - starvation fat mass ( patients with a higher initial fat mass gained more fat during recovery ) .
no patients in this study had fat mass increases that were sufficient to make them obese or to take them beyond the normal range for fat mass .
several studies reported that inclusion of resistance exercise training led to gains maintenance in lean body mass and better functional improvement results ( strawford et al 1998 , 1999 ) .
old formulation ma oral suspension ( megace os , bristol - myers squibb , princeton , nj , usa ) is best given at a dosage of 480800 mg with a maximum daily dose of 800 mg and taken with meals ( pascual lopez et al 2004 ; berenstein and ortiz 2005 ) .
newer ma nanocrystal oral suspension ( megace es ) employs a nanocrystal strategy that increases the surface area and , therefore , the absorption and bioavailability of the drug .
it can be given at a dosage of 625 mg ( 5ml ) daily in the fed or unfed state .
most patients treated with conventional ma suspension had improved appetite by 6 weeks , although weight gain was not yet significant at that time .
a course of treatment with ma for 12 weeks is probably enough to improve the appetite for eventual weight gain ( yeh et al 2000b ) .
lambert and co - workers found that despite significant weight gain , ma appears to have an anti - anabolic effect on muscle size even when combined with testosterone replacement .
resistance exercise attenuated this reduction in muscle mass . when ma is combined with testosterone and resistance exercise ,
resistance exercise had an anabolic effect on muscle mass ( lambert et al 2002 ) .
thus , patients should also receive strength and a local , muscular , endurance - training program to improve their functional status .
if there is no improvement in appetite by week eight , we suggest discontinuing the usage of ma because of the potential secondary adrenal suppression and adrenal insufficiency . common clinical practice has been to stop ma without tapering the dose because of its long half - life and storage in adipose tissue ( loprinzi 1996 ; loprinzi et al 1992a , 1992c ) .
no untoward clinical sequelae have been seen in most patients ( tchekmedyian et al 1987 , 1991 ; oster et al 1994 ; pascual lopez et al 2004 ; simmons et al 2004 ) .
the deposition of ma in fat stores may allow for a naturally occurring slow tapering of glucocorticoid activity after discontinuation of therapy .
patients who require more than 12 weeks of treatment should have their morning free cortisol levels checked at 12 weeks and biweekly thereafter .
a patient with adrenal suppression , ascertained by an abnormal adrenocorticotropic hormone ( acth ) stimulation test , but with no symptoms of adrenal insufficiency , should be monitored for fever , nausea and vomiting , hypotension , dehydration , clouded sensorium , pain in the abdomen , joints , and muscles , hypoglycemia , and acidosis . upon withdrawal of the drug , biochemical suppression of cortisol alone
if , on the other hand , patients have already developed a cushingoid appearance , abrupt discontinuation of ma could precipitate an addisonian crisis .
this can be prevented by supportive steroid therapy starting at a dosage of prednisone of 7.5 mg / day that is tapered at a rate of 2.5 mg / day every 23 weeks for patients who have recently been treated with a course of ma and have an abnormal acth level .
it appears reasonable to recommend that stress doses of prednisone be given during a period of acute illness or prior to surgery to prevent possible complications due to adrenal suppression .
the problem with ma oral suspension is its poorly solubility and absorption in a fasting state ( femia and goyette 2005 ) .
a recent study revealed that conventional ma suspension is best absorbed in the fed state .
its serum concentration and effect is many times higher when given in a fed rather than under fasting conditions ( alakhov et al 2004 ; femia and goyette 2005 ) .
newer ma nanocrystal oral suspension ( megace es ) can be given at a dosage of 625 mg ( 5 ml ) daily without meals .
there was a significant plasma concentration difference in fed versus fasting study beagle dogs ( femia and goyette 2005 ) .
a significant relationship was observed between weight gain and the percentage of the 24-hour administration interval , during which plasma concentrations of ma exceeded 300 ng / ml ( graham et al 1994 ) .
this may be the best explanation for the results in our clinical study , in which one third of the sickest study patients did not respond to ma oral suspension .
these non - responders were exactly the study patients who were too weak and anorexic to eat and who took the study medication in the fasting state ) ( yeh et al 2000b ) .
ma nanocrystalline particles significantly increased surface area per unit mass ( femia and goyette 2005 ) .
ma nanocrystalline particles increase the rate of absorption and decrease absorption variability when the drug is taken with food ( alakhov et al 2004 ; femia and goyette 2005 ) . ma
nanocrystal oral suspension ( megace es ) can be given at a dosage of 625 mg ( 5 ml ) daily without meals .
preclinical pharmacokinetic data suggest that the new ma formulation has the potential to significantly shorten the time to clinical response and thus may improve outcomes in patients with anorexia cachexia ( femia and goyette 2005 ) .
clinical studies in cancer , geriatric and hiv patients might be conducted to demonstrate the benefit of the neww formulation .
patients with documented involuntary weight loss should be screened for possible reversible causes and should also be checked for untreated hyperthyroidism , depression , poor dentition , uncontrolled diabetes mellitus , severe dysphagia , or malabsorption .
they should receive aggressive nutritional support ( food supplements [ 1.52 g / kg of protein daily , 2535 cal / kg daily ) , for a total of 2500 cal / day , dietitian consultations , and assistance with feeding ) . if other treatable etiologies are ruled out , ma should be considered for use as an appetite stimulant .
because all other anabolic agents such as growth hormone depend on adequate energy intake for optimal effect , the role of nutritional support and appetite stimulants remains fundamental .
patients will typically improve their appetite within the first few weeks after starting the drug .
resistance exercise traing coupled with the weight gain may lead to improved lean muscle mass .
the newer ma nanocrystal oral suspension ( megace es ) can be given in a smaller volume and depends less on a patient taking it in the fed state to achieve a clinical benefit . | the aim is to review major clinical trials that have used megestrol acetate ( ma ) in the treatment of cachexia across several disease states .
a review of general usage and potential side - effects are discussed . a theory that the newly approved nanocrystal formation of ma can better deliver this potent medication for treatment
will also be reviewed . |
cumulative trauma disorders ( ctds ) are defined as muscle , nerve , tendon , joint , cartilage ,
and spinal disc damages or disorders due to exposure to risk factors in the work
environment1 .
the common characteristics
of ctds are that they arise or increase due to working and that they may lead to limitations
on activities in the business environment or nonbusiness activities2 .
these disorders , which do not include injuries due to
accidents , generally develop in the musculoskeletal system over a long term1 .
ctds are basically classified into two
groups as upper extremity disorders ( ue - ctd ) and waist disorders .
these disorders create
negative effects in business life on productivity , costs , and the quality of the lives of
employees .
the frequency of ue - ctd is getting higher , and because the losses of labor it
causes , economic losses , and decreases in quality of life of employees , it has become a
serious public health problem3 . a doner kebab chef or
doner chef is a person with the skills needed to marinate the meats
to be used in a doner kebab , place a doner in a cooker , cook it , cut it in leaf form , and
serve it in the desired portions .
the job of doner chef is specific to turkey , and their
upper extremities are used in performance of this business .
especially when cutting a doner ,
they frequently repeatedly perform upper shoulder flexion and extension and wrist flexion
and extension movements .
the doner knife , which is longer and heavier than a normal life ,
makes this work harder , and this leads to problems in the elbows and wrists . in this study ,
we examined the frequency of ue - ctd in doner chefs , and whether it increases in comparison
with individuals who do not need to perform repetitive , forceful upper extremity movements
in their jobs .
volunteers who did not need to perform repetitive , forceful upper extremity movements in
their jobs and doner chefs were selected for this study . those having systemic diseases
( diabetes , rheumatic diseases , muscle diseases , neuropathy , etc . ) leading to upper extremity
problem or congenital or acquired upper extremity damages ( deformity , torticollis ,
scoliosis , loss of sense , nerve damage , etc . )
all of the
participants were informed about the study , and written consent was obtained from them .
face - to - face interviews were performed by visiting the cases in their workplaces .
a survey
form containing questions about demographic information , age , occupational experience
( years ) , daily working duration , height , weight , any second job , and diseases was provided .
all of the
participants were also asked about any musculoskeletal system complaints ( lasted for at
least 1 week ) in last 6 months , and both of their upper extremity musculoskeletal systems
were physically examined . in the physical examination ,
the subjects were examined for
deformity , color change , panicula , atrophy , and hypertrophy through inspection of the neck
and upper extremity .
temperature increase and painful points were investigated through
palpation of bones , joints , and soft tissues .
measuring the active - passive movement gap of
all of the joints , the subjects were examined for any limitation , crepitation , or
mobility - induced pain .
the manual muscle test and a neurologic examination for the upper
extremity were carried out .
then a definitive diagnosis was made via special tests specific
to upper extremity problems .
the special tests performed were the spurling test , neer test
( subacromial impingement test ) , speed test , cozen test ( tennis elbow test ) , golfer s elbow
test , finkelstein test , tinel test , and phalen test4,5,6,7,8 .
statistical assessments were carried out by using the statistical package for social
sciences ( spss , inc . , chicago , il , usa ) 16.0 software . besides the definitive statistical
methods ( average , standard deviation ) during assessment , intergroup comparisons of age ,
experience ( years ) , and daily work duration were carried out via the independent t test ,
while comparisons of complaint and disorder rates were carried out via the
test .
results were accepted as
statistically significant if the p value was lower than 0.05 .
eighty - two individuals working as doner chefs and 82 individuals not working as doner chefs
participated into the study voluntarily .
all of the participants were male , and there was no
difference among groups in terms of age , experience ( years ) , daily work hours , and body mass
index ( table 1table 1.comparison of age , bmi and duration of work between the two groupsstudy group(n=82)control group(n=82)age34.466.75328.86bmi26.052.9426.084.78work experience ( years)13.055.4610.408.71duration of work ( hours / day)9.891.7610.481.75 ) .
upper extremity musculoskeletal system complaints were detected in 44.8% of the study group
and 34.7% of the control group , while upper extremity musculoskeletal system disorders have
been detected in 20.6% of the study group and 15.6% of the control group .
there was no
statistical difference among the two groups in terms of rates of upper extremity
musculoskeletal system complaints and disorders ( p>0.05 ) .
the doner chefs having lateral epicondylitis had 17.668.01 years of work experience , while
doner chefs not having lateral epicondylitis had 10.408.71 years of work experience
( p=0.005 ) .
comparisons of groups in terms of the upper extremity complaints and in terms of
the diagnosis of the upper limbs are presented in tables 2 and 3table 2.comparison of upper extremity complaints between groupsstudy group(n=82)control group(n=82)hand pain10 ( % 12)1 ( 1.2%)hand numbness1 ( % 1.2)1 ( 1.2%)elbow pain13 ( % 15.8)4 ( 4.8%)shoulder pain7 ( % 8.5)7 ( 8.5%)neck pain6 ( % 7.3)11 ( % 13)table 3.comparison of diagnosis of the upper limbs between groupsstudy group(n=82)control group(n=82)carpal tunnel syndrome1 ( 1.2%)1 ( 1.2%)de quervain s disease1 ( 1.2%)3 ( 3.6%)lateral epicondylitis12 ( 14.6%)2 ( 2.4%)medial epicondylitis1 ( 1.2%)2 ( 2.4%)biceps tendinitis1 ( 1.2%)0 ( 0%)rotator cuff tear1 ( 1.2%)0 ( 0% ) .
one of the most important complaints of working people is work - related upper extremity
disorders ( ueds)9 .
the main complaint in
ue - ctd is pain in the upper extremities , neck , shoulder , elbows , wrists , and waist .
exclusive usage of extensor muscles leads to lateral epicondylitis , and two of its results
are inflammation and irritation of the tendon insertion10 .
one of the most important results of our study is that lateral epicondylitis occurs less
frequently in doner chefs than in other members of society . also , the working durations of
doner chefs with lateral epicondylitis were longer than in those not having lateral
epicondylitis .
lateral epicondylitis is seen frequently in people working in meat factories
( 6.4% ) , sausage makers ( 11.3% ) , packers ( 7% ) , nursery school cooks ( 10.5% ) , and people
working in manufacturing ( 5.8% ) , fish processing ( 14.3% ) , and textile production . the
frequency of lateral epicondylitis increases as the exposure duration increases11,12,13,14 .
the rate of lateral epicondylitis was 14.6% in doner chefs in our study , while the rate of
hand pain was 12% .
the frequency of lateral epicondylitis or tennis elbow varies between 1%
and 3% among the general population15 ,
while it varies between 2% and 23% among the working population16 .
it has been revealed in earlier epidemiologic articles
that lateral epicondylitis occurs due to forceful movements of the elbow17,18,19 . on the other hand , the results reported
on the relationship between lateral epicondylitis and arm movement are inconsistent20 .
epicondylitis risk was defined by job title in a review by palmer et al21 . on the other hand , in a more recent
article ,
the
studies that reported only results concerning epicondylitis were excluded from that article .
palmer et al . found that the risk of epicondylitis significantly increases in working people
exposed to vibration , such as workers , foresters , pipe fitters and water / gas suppliers , meat
cutters , packers , and japanese nursery school cooks . in parallel with these findings ,
the
findings of the present study indicate an increased lateral epicondylitis risk in male meat
cutters .
the present study improves the existing knowledge by providing quantitative
information for exposure - response relationships between work - related factors and the
occurrence of four specific elbow disorders .
but many different exposure studies and
different diagnoses for specific elbow complaints have been reported , and so study pooling
is not suitable in this field .
when the employees are exposed to combined risk factors , such as force and repetition , or
either force or repetition and extreme non - neutral arm postures , the relationship between
workload factors and epicondylitis reaches its maximum point11 , 22 .
we have revealed that
lateral epicondylitis is significantly related to work tasks requiring a combination of
repetitive and forceful activities and also with a longer exposure durations .
the exposure to physical loads during work is generally evaluated and investigated by
self - report in epidemiologic articles .
this method has the advantage of affording the
possibility to study cumulative exposure over time , but its reliability or validity may be
questionable23 .
the most difficult
parts are the assessments of the weights of handled loads and the exerted forces .
the revealed relationships were found to be stronger among physical factors rather than
psychosocial ones .
low social support , one of the psychosocial factors , remains significant
only for men , even after adjustment , and other factors became nonsignificant for both
genders , but some other authors have revealed significant associations between psychosocial
factors and elbow disorders24 , 25 . for assessing the links between psychosocial factors and
physical ones , further research is required26 , 27 . the monthly income level and their
educational statuses are low .
the frequency of upper extremity tendinitis has been found to be higher in obese
subjects28 . in our study , the bmis of
doner chefs classified them as obese .
it has been shown in a cross - sectional study that the effect of aging can not be
separated from the cumulative effect of current and earlier deleterious exposure . on the
other hand , in parallel with the theory about the aging affect associated with tissue
degeneration29 , in the present study ,
the effect of age remained significant among employees working in the same job for more than
10 years .
as stated in the previous articles about elbow disorder30 , 31 , elbow pain and
epicondylitis are associated with other musculoskeletal disorders .
this reveals the
complexity of movements and consequently the pain in different body parts . because the mean
age of our study group was 34.466.75 years
, our 12 subjects with lateral epicondylitis
cases were advanced - aged patients . in conclusion , our study shows that lateral epicondylitis in the elbow is a common
work - related upper limb disorders among doner chefs .
low socioeconomic level , age , obesity ,
and complex hand - wrist movements increase this risk . in occupational groups such as doner chefs
, employees should not perform the same tasks
repetitively , the durations of breaks should be modified , and wearing splints should be
ensured if possible to decrease the risk of lateral epicondylitis . | [ purpose ] doner kebab is a food specific to turkey ; it is a cone - shaped meat placed
vertically on a high stand .
the doner kebab chefs stand against the meat and cut it by
using both of their upper extremities .
this work style may lead to recurrent trauma and
correspondingly the upper extremity problems .
the aim of this study was to investigate the
upper extremity disorders of doner chefs .
[ subjects and methods ] doner kebab chefs were
selected as the study group , and volunteers who were not doner kebab chefs and did nt
exert intense effort with upper extremities their business lives were selected as the
control group .
a survey form was prepared to obtain data about the participants ages ,
working experience ( years ) , daily work hours , work at a second job , diseases , drug usage ,
and any musculoskeletal ( lasting at least 1 week ) complaint in last 6 months .
[ results ] a
total of 164 individuals participated in the study , 82 doner chefs and 82 volunteers . in
20.6% of the study group and 15.6% of the control group ,
an upper extremity
musculoskeletal system disorder was detected .
lateral epicondylitis was more frequently
statistically significant in the work group .
[ conclusion ] hand pain and lateral
epicondylitis are more frequent in doner chefs than in other forms of business . |
alzheimer 's disease ( ad ) is a multifactorial disorder leading to progressive memory loss and eventually death .
one of the histopathological hallmarks of ad is the excessive accumulation of amyloid beta ( a ) peptides in the brain resulting in extracellular amyloid plaques [ 13 ] .
amyloid peptides are natural cleavage products derived from the amyloid precursor protein ( app ) via sequential endoproteolytic processing operated by specific secretases , beta - site amyloid precursor protein cleaving enzyme 1 ( bace-1 ) , and -secretase .
length of a peptides ranges from 36 to 43 amino acids . while a140 and a142 represent the predominant isoform , abundance of a140 is generally higher compared to a142 which is prone to aggregate and exhibits stronger cytotoxicity .
app mutations can be proamyloidogenic facilitating the generation of cytotoxic a peptides . for instance , the swedish double mutation km670/671nl promotes bace cleavage and increases abundance of both a140 and a142 whereas mutations at amino acid 717 augment generation of the more toxic a142 .
presenilin- ( ps ) 1 and presenilin-2 act as catalytic sites for -secretase and mutations in ps , which are much more common than app mutations , further enhance the production of proamyloidogenic a142 .
besides the formation of a plaques , human ad neurons also present neurofibrillary tangles , that is , intraneuronal inclusions of hyperphosphorylated tau ( ) protein [ 9 , 10 ] .
numerous transgenic mouse models of ad exist that are mainly characterized by overexpression of one or more mutant apps resulting in excessive amounts of a142 .
the latter causes age - related ad - specific abnormalities including a plaques , axonal and synaptic dystrophy , impaired synaptic plasticity , and impaired learning and memory [ 1114 ] . in this study
an appsweps1de ad mouse model has been used which is characterized by carrying human app with swedish double mutation ( appswe ) cointegrated with human ps-1 with exon 9 deletion ( ps1de9 ) [ 1517 ] .
this leads to overproduction of app and ps1 splice variants with subsequent increase in neural a load .
about 100 ad mouse models have been established so far and they differ significantly in pathohistomorphology , symptomatology , disease progression , mortality , and translational categories such as isomorphism , homology , and predictability . in general , transgenic ad lines display a142 overload and this might be related to increase in mortality and death with the latter occurring out of a sudden in most cases [ 1921 ] . in the appsweps1de9 line
appsweps1de9 mice develop first a plaques around 4 months of age , particularly in the cortex and hippocampus .
this coincides with a mortality peak around 3 - 4 months of age [ 23 , 24 ] . at the age of 6 months memory deficits in radial arm water maze
are prominent whereas , at 12 months , mice start exhibiting behavioral and cognitive deficits detectable in spatial navigation , reference learning , and morris water maze .
it has been hypothesized that seizure activity might account for sudden death in this line as intracerebral a accumulation was suggested to be causally linked to epileptogenesis [ 26 , 27 ] .
in addition , neurodegenerative processes in the cortex and septohippocampal system can result in complex central dysrhythmia particularly affecting theta and gamma activity .
previous studies have analyzed selective electrical activity and individual frequency characteristics from electrocorticograms and other deflections including sleep studies in app transgenic mice [ 4 , 22 , 26 , 2931 ] .
in contrast to these studies , we present a systematic fft based frequency analysis and multiparameter longitudinal seizure evaluation from both cortical m1 and hippocampal ca1 recordings in appsweps1de9 and controls based on nonrestraining eeg radiotelemetry .
also for the first time we perform gender - specific analysis that reveals striking age - dependent differences in theta / gamma activity between male and female ad mice gaining further insight into gender - specific differences in the pathogenesis and symptomatology of ad .
this study was performed in appsweps1de9 transgenic mice with a c57bl/6j background carrying a human app with swedish double mutation ( appswe ) cointegrated with human ps1 with exon 9 deletion ( ps1de ) [ 15 , 16 ] .
34832-jax ) were purchased from jackson laboratory . in total , 21 wt controls ( 10 males , body weight : 26.94 0.64 g ; 11 females , body weight : 21.23 0.53 g ) and 20 appsweps1de mice ( 9 males , body weight : 26.16 0.56 g ; 11 females , body weight : 21.73 0.35 g ) were analysed in this study separately . body weight was determined at 14 wks of age .
importantly , both genders were used and analysed in this study . due to the long - term experimental character and experimental time schedules ( see the following ) , females could not be recorded within the same stage of the estrous cycle . in addition , the stages of the estrous cycle were reported not to influence basic eeg related parameters .
all experimental mice were housed in groups of 3 - 4 in clear makrolon cages type ii with ad libitum access to drinking water and standard food pellets .
mice were maintained at a temperature of 21 2c , 5060% relative humidity , and on a conventional 12 h/12 h light / dark cycle beginning at 5:00 am using ventilated cabinets ( model 9av125p ) .
subsequent epidural , that is , electrocorticographic and deep , intracerebral , that is , electrohippocampographic , recordings were performed inside the ventilated cabinets .
all animal experimentation was performed according to the guidelines of the german council on animal care and all protocols were approved by the local institutional and national committee on animal care ( landesamt fr natur , umwelt und verbraucherschutz , lanuv , germany ) .
the authors further certify that all animal experimentation was carried out in accordance with the european communities council directive of november 24 , 1986 ( 86/609/eec ) and of september 22 , 2010 ( 2010/63/eu ) .
special attention was paid to minimize the animal sample size and the suffering of mice .
mice were anesthetized using the inhalation narcotic isoflurane ( baxter 100% v / v ) .
the volatile anesthetic was applied via facemask using a matrix tm vip 3000 calibrated vaporizer and a scavenger system from harvard apparatus ( usa ) as described previously [ 3234 ] .
the radiofrequency transmitter tl11m2-f20-eet ( 2-channel transmitter , data science international ( dsi , germany ) ( specifications : weight 3.9 g , volume 1.9 cc , input voltage range 1.25 mv , amplification factor ( voltage gain ) 200 , and nominal sampling rate 250 hz ) was implanted into a subcutaneous pouch on the back of the experimental animals .
the eeg electrodes of both radiotelemetry transmitter channels were stereotaxically positioned via a computerized 3d stereotaxic stereodrive system ( neurostar , germany ) [ 3234 ] .
the differential epidural surface electrode of channel 1 of the tl11m2-f20-eet transmitter was positioned at the following stereotaxic coordinates referring to the bregma craniometric landmark : ( + ) -lead , cranial + 1 mm , and lateral of bregma 1.5 mm ( left hemisphere ) .
an epidural reference electrode was placed on the cerebellar cortex at bregma 6 mm , lateral of bregma 1 mm ( left hemisphere ) .
note that for epidural electrode placement the sensing lead of the transmitter is bent 90 and directly placed on the surface of the cortex . for deep , intracerebral eeg recordings targeting the hippocampal ca1 region ,
the differential electrode of channel 2 of the tl11m2-f20-eet transmitter was positioned as follows : ( + ) -lead , caudal 2 mm , lateral of bregma 1.5 mm ( right hemisphere ) , and dorsoventral ( depth ) 1.5 mm .
an epidural reference electrode was placed on the cerebellar cortex at bregma 6 mm and lateral of bregma 1 mm ( right hemisphere ) . for deep recordings ,
the deep tungsten electrodes ( fhc , bowdoin , usa ) are insulated with epoxylite with a shank diameter of 250 m and an impedance of 50100 k ( measured at 1000 hz ) .
surface and deep electrodes were fixed using glass ionomer cement ( kent dental , uk ) and the scalp was closed using over - and - over sutures ( ethilon , 6 - 0 ) .
as mice are predisposed to hypothermia , supplemental warmth was given to the animal with a heating pad during the whole surgical procedure .
a detailed description of the stereotaxic electrode placement and transmitter implantation was previously described in detail by weiergrber and colleagues [ 3234 ] .
carprofen ( 5 mg / kg , rimadyl , parke - davis / pfizer , germany ) was administered subcutaneously for postoperative pain management .
this recovery period is based on the observation that no differences in basic physiological / behavioral parameters such as food and water uptake , motor activity , and body temperature could be detected between radiotransmitter implanted , nonimplanted , and sham - operated mice 10 days after surgery . to verify whether electrodes were placed in the correct target region , brains were extirpated postmortem and fixed in 4% paraformaldehyde .
subsequently , brains were cut to 60 m slices using a vibroslice tissue cutter ems 5000-mz ( campden instruments limited , uk ) .
animals , in which electrodes turned out to be not in the exact target position , were excluded from analysis .
the first long - term recording of 48 hrs was performed at day 10 after surgery from both the primary motor cortex ( m1 ) and the ca1 hippocampal region . a second 48 hrs long - term recording
was performed at day 17 after implantation using both deflections . for eeg data acquisition , the dataquest art 4.2 software ( dsi ) was used .
the nominal sampling rate ( f ) of the tl11m2-f20-eet transmitter is 250 hz . the virtual interpolated sampling rate was manually adjusted in the dataquest art 4.2 configuration window to 500 hz per channel for better visualisation .
note that analysis was performed up to 70 hz , considering the transmitter specific bandwidth and the nyquist - shannon limit of 125 hz for this transmitter type .
as the animal moves about in its cage , the telemetry signal transmitted to the receiver antennas varies in strength .
the signal strength may vary due to orientation of the animal relative to the receiver or due to the distance of the animal from the receiver antennas . when the signal strength changes by a certain amount , an activity count is generated .
the number of counts generated is dependent on both distance and speed of movement ( acceleration ) .
note that the activity data provided by dataquest art is a relative measure of locomotor activity .
activity analysis was carried out for controls and appsweps1de9 mice from both genders . for detailed analysis data
qualitative and quantitative seizure analysis was carried out using the neuroscore seizure detection and quantification module .
as the seizure detection module requests configuration of the detection tool , spike detection characteristics including absolute threshold , spike duration limits , and spike interval limits were adapted for different seizure protocols . using the absolute threshold approach for ictal discharge detection , the maximum amplitude was set as 1000 v with an average spike detection threshold value of 200 v . in both seizure detection protocols the minimum spike duration
was determined as 1 ms and the maximum spike and short / slow - wave duration at 100 ms .
spike trains were detected with a minimum train duration of 0.5 s including at least 4 individual spikes .
spike intervals within a train were ranging between 0.05 and 0.3 s. the interval between individual spike trains was determined at 1 s. application of seizure protocols resulted in total number of seizure episodes and spikes , spike frequency , total spike train duration , and shortest and longest spike train duration .
significance was calculated using student 's t - test which included pretesting for normal distribution via the kolmogorov - smirnov test .
all statistics and graph presentation in this section were performed with graphpad prism 6 for windows .
recordings ( 48 hrs ) of spontaneous eeg activity were performed based on a nominal sampling rate of 250 hz of the radiofrequency transmitter and a virtual ( software based ) sampling rate of 500 hz ( per channel ) .
eeg data were fft analyzed using neuroscore 2.1 ( dsi ) in the frequency range of 0.570 hz , thus comprising the typical delta ( 0.54 hz ) , theta ( 48 hz ) , beta ( 1230 hz ) , and gamma bands of 3050 hz and 5070 hz . the upper gamma limit ( 70 hz ) is still below the nyquist - shannon limit of 125 hz ; thus fft based analysis is possible .
the length of the individual eeg epochs that were fft analyzed was 2 s. subsequently , mean eeg power [ % ] was calculated for the individual frequency ranges , for both genders and the individual circadian stages , that is , two dark ( d1 , d2 ) and two light cycles ( l1 , l2 ) .
in addition , activity data of mice during the conventional 12 h light / dark cycle ( starting at 5:00 a.m. ) were used to correlate activity in different eeg frequency bands from both deflections with either the active ( activity units > 0 ) or inactive state ( activity units = 0 ) .
statistics for frequency analysis were carried out using multiple student 's t - test , corrected for multiple comparison using the holm - sidak method ( p < 0.05 ; p < 0.01 ; p < 0.001 ) .
most of the statistics and graph presentations were performed with graphpad prism 6 for windows .
as some alzheimer mouse lines were reported to exhibit developmental deficits , we closely monitored the gender - specific body weight development of appsweps1de9 and control mice . in age - matched transgenic and control mice it turned out that males exhibited significant higher body weight compared to females .
however , different from other alzheimer mouse models such as the 5xfad [ 27 , 28 ] no significant reduction of body weight in appsweps1de9 could be detected compared to controls in our study at 14 wks of age ( control males 26.94 0.64 g ( n = 10 ) versus transgenic males 26.16 0.56 g ( n = 9 ) , p = 0.3755 ; control females 21.23 0.53 g ( n = 11 ) versus transgenic females 21.73 0.35 g ( n = 11 ) , p = 0.4416 ) . the 48 hrs ca1 and m1 long - term eeg recordings at the age of 14 , 15 , 18 , and 19 weeks from both control mice and appsweps1de9 animals
as illustrated in figures 1(a)1(d ) , appsweps1de9 exhibited typical ictal like discharges , including spikes , spike - waves , and polyspike waves . despite isolated ictal discharges we could also observe characteristic spike / spike - wave trains including the seizure initiation phase , seizure continuation , and termination ( figures 1(a)1(d ) ) .
as reported previously , seizures often began with a large - amplitude spike and subsequent spike trains were typically , however not always followed by so - called postictal depression ( figures 1(a)1(d ) ) .
abnormal electroencephalographic ictal discharges were present in both ca1 and m1 deflections from both genders . in controls ,
ictal discharges were hardly detected and could be related to behavioral phenomena , for example , eating and grooming . in this study , we did not analyze motoric exacerbation of aberrant hyperexcitability , that is , behavioral seizure activity , but strictly focused on electroencephalographic seizure activity .
we analyzed motor activity in controls and appsweps1de9 mice as both seizure activity and frequency characteristics can be associated or modified by motoric behavior ( figure 2 ) .
the activity was calculated for the first ( d1 ) and second ( d2 ) dark cycle ( of each 48 hrs recording ) and for the total dark period ( d1 + d2 ) .
the same holds true for the two light cycles ( l1 , l2 of each 48 hrs recording ) as well as the total light period ( l1 + l2 ) . in females , no significant difference in motor activity could be detected between controls and appsweps1de9 mice at any circadian stage ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7167358 ) . in males , a significant increase in relative motor activity was observed during the dark cycle ( d1 , 5089.50 1504.47 versus 10983.50 2567.92 , p = 0.0412 ; d2 , 3700.25 909.43 versus 9994.00 2333.55 , p = 0.0049 ; d1 + d2 , 8789.75 2410.34 versus 20977.50 4812.86 , p = 0.0127 ) at the age of 18 wks ( figures 2(a ) , 2(c ) , and 2(e ) ) . however , no additional alteration in motor activity could be detected in males at any other time point . in order to quantify electroencephalographic seizure activity in controls and appsweps1de9 mice we used an automatic seizure detection tool .
this tool was applied for the individual circadian stages , that is , the dark ( d ) and light ( l ) stage , and various seizure parameters were evaluated , including the number of spike trains , total spike train duration , average spike train duration , and number of single spikes .
these parameters were calculated again for both ca1 and m1 deflections in both genders . as it has been reported for other alzheimer mouse models as well
[ 26 , 28 ] , appsweps1de9 mice exhibit a high variability in seizure severity ( figure 3 ) . significant increases in spike / spike train parameters were detected consistently in m1 and ca1 deflections from both genders .
note that overall electroencephalographic seizure activity in males obtained from m1 and ca1 is most prominent at an age of 14 - 15 wks ( figures 3(a)(i , iii , v , and vii ) and 3(b)(i , iii , v , and vii ) ) whereas related activity in females is also present at 18 - 19 wks of age ( figures 3(a)(ii , iv , vi , and viii ) , and 3(b)(ii , iv , vi , and viii ) ; see also supplementary figure 2 ) .
this observation clearly correlates with proven age- and gender - specific differences in plaques formation in appsweps1de9 mice . in male appsweps1de9 mice a significant reduction in ca1 relative theta power
was observed at the age of 14 wks ( dark cycle : 28.890 0.902 versus 20.978 2.125 , p = 0.0052 ; light cycle : 29.101 0.754 versus 23.578
2.091 , p = 0.0253 ) but not later ( figures 4(a)(i ) and 4(b)(i ) ) .
however , this phenomenon occurred at later stage ( 18 wks , dark cycle : 22.528 1.485 versus 14.870 3.136 , p = 0.0495 ; light cycle : 24.394 1.201 versus 17.487 2.484 , p = 0.0313 ) and at 19 wks of age ( dark cycle : 22.898 2.458 versus 14.245 3.002 , p = 0.0673 ; light cycle : 24.691 1.895 versus 16.084 2.994 , p = 0.0512 ) ( figures 4(a)(iv ) and 4(b)(iv ) ) .
these findings clearly point to a circadian independent alteration in theta architecture and the developmental time - dependent characteristics of theta are gender - specific .
in contrast to other alzheimer mouse models that exhibit an increase in theta activity [ 28 , 38 ] , the appsweps1de9 model presents age - specific reduction in theta power that is in line with many other ad mouse lines [ 3941 ] .
interestingly , the age - specific theta activity pattern in both genders is mirrored in the gamma activity in the ca1 deflection as well .
male appsweps1de9 mice exhibit an increase in low gamma ( 3050 hz ) at early age of 14 wks ( dark cycle : 6.556 0.298 versus 8.713 0.992 , p = 0.0502 ; light cycle : 5.138 0.296 versus 8.154 1.367 , p = 0.0424 , figures 4(a)(ii ) and 4(b)(ii ) ) which is not observed in females .
in contrast , females display significant increase in low gamma ( 3050 hz ) at the age of 18 wks ( dark cycle : 8.083 1.314 versus 11.114 1.250 , p = 0.1455 ; light cycle : 3.406 0.583 versus 10.611 1.051 , p < 0.001 ) and 19 wks ( dark cycle : 6.399 1.633 versus 11.886 1.236 , p = 0.0366 ; light cycle : 5.468 1.065 versus 11.045 1.836 , p = 0.0391 ) ( figures 4(a)(v ) and 4(b)(v ) ) .
male appsweps1de9 again showed increased activity at 14 wks of age ( dark cycle : 3.540 0.182 versus 4.956 0.649 , p = 0.0481 ; light cycle : 2.508 0.189 versus 4.193 0.834 , p = 0.0593 , figures 4(a)(iii ) and 4(b)(iii ) ) , whereas females presented an increase at later stages , that is , 18 wks ( dark cycle : 4.351 0.787 versus 8.273 1.379 , p = 0.0351 ; light cycle : 3.572 0.636 versus 7.038 1.146 , p = 0.0266 ) and 19 wks ( dark cycle : 3.556 1.159 versus 8.128 1.245 , p = 0.0362 ; light cycle : 2.981 0.845 versus 7.265 1.441 , p = 0.0427 ) ( figures 4(a)(vi ) and 4(b)(vi ) ) .
note that analysis of higher gamma frequency is not recommendable / possible given the transmitter bandwidth of 150 hz and the nyquist - shannon limit of the transmitter of 125 hz . in summary , theta activity decrease and gamma activity increase
we further tested this hypothesis with a univariate two - way anova with between - subjects factors ( i ) genotype , that is , controls and appsweps1de9 , and ( ii ) frequency band , that is , theta and gamma band power for ca1 .
based on the previous statistical results , we choose data of 14-week - old male mice and 18-week - old female mice . for each gender , the subgamma band with the highest p value was selected .
it turned out that this results in a cycle - dependent difference , but not a gender - dependent difference ( dark cycle : higher gamma band ( 5070 hz ) for both male and female ; light cycle : lower gamma band ( 3050 hz ) for both male and female ) . based on this procedure ,
the statistical computations lead to a significant interaction between factor ( i ) genotype and factor ( ii ) frequency band for all four constellations ( males , dark cycle : p = 0.0006 ; males , light cycle : p = 0.0083 ; females , dark cycle : p = 0.0058 ; females , light cycle : p = 0.0002 ) , which demonstrates a clear connection between the decrease of theta power and the increase of gamma power .
in addition to the gender - specific fft based eeg power analysis we further checked for activity dependent power alterations between the active and inactive state ( supplementary figure 3 ) . during the dark cycle , it turned out that significant changes in males could not be detected . in females , the observed changes ( figure 4 ) persisted , particularly in the inactive state .
this held true for the theta and both gamma frequency ranges analyzed . in the light phase , males displayed only minor changes in the theta range , but not the gamma ranges .
females , however , again exhibited prominent alterations in theta and both gamma ranges predominantly in the inactive stage .
the antithetic distribution of reduced theta and increased gamma power in females was most outstanding during the inactive state ( supplementary figure 3 ) .
we also performed fft based eeg power analysis for m1 recordings from controls and appsweps1de9 .
no changes were observed for the beta frequency range ( 1630 hz , figure 4(c ) ) . for the delta range ( 0.54 hz ) , there turned out to be only a significant reduction in relative power in males during light cycle at the age of 18 wks ( 30.469 2.189 versus 18.426 2.876 , p = 0.0158 ) .
alzheimer 's disease is a neurodegenerative disorder that is accompanied by neural cell loss that ultimately results in neural network dysfunction , such as dysrhythmia and/or aberrant excitability . in humans
, ad goes together with an increase of prevalence of spontaneous , unprovoked seizures in the elderly [ 4245 ] . within the last years
there is increasing evidence that this phenomenon also holds true for various ad mouse models [ 22 , 2628 , 31 ] .
it is assumed that a is a crucial trigger of ictogenesis / epileptogenesis in ad mice .
this is further supported by the observation that exogenous administration of a142 can lead to neural hyperexcitability in cortical and hippocampal slices of wt mice .
whether a plaques or high a concentrations exhibit a higher proictogenic potential has not been strictly proven yet .
importantly , seizures tend to peak around an age when amyloid plaques occur in both the cortex and hippocampus .
some previous studies have analyzed spontaneous electrical activity in electrocorticograms and carried out sleep studies in app transgenic mice [ 4 , 22 , 23 , 26 , 29 ] . unlike our nonrestraining eeg radiotelemetry approach , minkeviciene et al .
performed eeg recordings in appsweps1de9 mice from the frontal cortex using a restraining tethered system . whereas minkeviciene et al .
checked for the occurrence and duration of behavioral and electroencephalographic seizures , we performed detailed longitudinal analysis of multiple seizure parameters , such as number of spike trains , total spike train duration , average spike train duration , and total number of spikes . as it is known that gender and the estrous cycle in females in particular significantly affect seizure susceptibility / severity in rodents but also in humans [ 4648 ] , we performed for the first time an entire gender - specific seizure analysis of cortical m1 and hippocampal ca1 recordings ( figure 3 ) .
the electroencephalographic seizures exhibit typical appearance with an initial spike followed by a complex spike train and subsequent postictal depression following seizure termination ( figures 1(a)(ii ) , 1(b)(ii ) , 1(c)(ii ) , and 1(d)(ii ) ) .
in contrast to other reports [ 21 , 22 , 26 , 49 ] , no appsweps1de9 mouse died during the time course of our experiments , particularly not from spontaneous prolonged status epilepticus as has been suggested previously .
in contrast to earlier studies that neglected a potential gender - specific interference , our results illustrate complex age- and sex - dependent seizure architecture in appsweps1de9 mice . there is an ongoing discussion on how neural degeneration can actually evolve in aberrant excitability in neural networks , such as the septohippocampal circuitry .
there have been reports about loss of calbindin - positive immunoreactivity in dentate gyrus and abnormal npy immunoreactivity in mossy fiber projections to ca3 [ 22 , 26 ] with both alterations being interpreted as compensatory network changes .
studies by further suggest that hyperactive neuronal clusters surrounding amyloid plaques in the cortex of app / ps1 mice are involved in triggering seizures .
in addition , it has been hypothesized that in the course of a plaque formation disruption of voltage - gated ion channels [ 5155 ] and plasma membrane potential destabilisation might result in seizure activity .
in addition to seizure analysis , we also had a detailed fft based look at the cortical and hippocampal frequency characteristics , particularly the theta and gamma frequency range .
hippocampal theta rhythm is related to specific behavioral and cognitive processes including alertness , learning and memory , spatial navigation , and spatial memory . as the septohippocampal system
is pathophysiologically affected in ad , one might expect an impairment in hippocampal rhythmicity , particularly in the theta frequency range [ 5658 ] .
previous studies seemed to confirm this hypothesis showing that cognitive decline in ad is accompanied by decrease in theta activity [ 39 , 56 , 59 ] , whereas others report an increase in theta activity [ 28 , 5658 ] .
this paradoxical situation might be explainable given the sophisticated pathophysiology of the septohippocampal system and the heterogeneity , that is , dualistic theory of hippocampal theta activity , that is , hippocampal theta being differentiated into atropine - insensitive type i and atropine - sensitive type ii theta [ 40 , 60 ] .
it has been reported that a can modulate cholinergic and glutamatergic neurons in the septohippocampal system [ 6164 ] and that the presence of a in both the medial septum and the hippocampus can dampen theta rhythm in vitro and in vivo [ 62 , 6569 ] which is associated with cognitive impairment .
scott et al . reported about a decline in theta power at an age of 4 and 8 months in appsweps1de9 mice which was recorded under urethane anaesthesia and elicited by brainstem stimulation .
described alterations in local oscillatory activity in the theta range ; however these findings were obtained under restrained tether recording conditions using mixed , unbalanced gender distribution in the study groups which is critical for interpretation .
importantly , our study now clearly demonstrates under physiological , that is , spontaneous , conscious , and unrestrained , long - term recording conditions a reduction in theta power and a concomitant increase in gamma power that was not only age - dependent but also gender - specific ( figure 4 ) . whereas males exhibited a reduction in theta at an age of 14 wks that vanished while getting older , females exhibited theta power reduction at later stage of 18 and 19 wks of age . to our knowledge
this is the first time that age- and gender - specific alterations in hippocampal theta and gamma architecture have been described in an ad mouse model .
importantly , an activity dependent analysis confirmed the antithetic theta - gamma power distribution particularly in females .
it should be noted , that based on the dualistic theory , theta has to be differentiated into atropine - insensitive type i theta and atropine - sensitive type ii theta .
active exploratory behavior , for example , is normally associated with type i theta , whereas type ii theta is characteristic of alert immobility [ 26 , 28 , 67 , 69 , 71 , 72 ] .
disruption of theta activity results in spatial memory deficits , whereas the restoration of theta - like rhythmicity restores learning capabilities in rats .
we conclude that inactive mice in both the dark and light phase predominately exhibit alert immobility which is accompanied by atropine - sensitive type ii theta [ 74 , 75 ] .
thus , our data suggest that female appsweps1de9 mice in particular demonstrate reduced atropine - sensitive type ii theta at later stages which is likely to be due to septohippocampal impairment during ad development .
previously , hippocampal in vitro preparations from 1-month - old tgcrnd8 ad mice were shown to exhibit robust alterations in theta - gamma coupling although these mice expressed negligible amounts of a .
in contrast to our study , goutagny et al . did not observe changes in theta or low gamma power in the tgcrnd8 model of ad .
interestingly , rubio et al . described a decrease in both hippocampal theta and gamma power in happ ad mice which is antithetic to our results .
it is commonly accepted that , in the appsweps1de9 mouse model , a burden of aged females appears to be more severe than in old males [ 23 , 7779 ] .
however , examination of a dynamics in this particular mouse line revealed additional age- and gender - related specificities . in 4-month - old appsweps1de9 mice ,
brain a load has been reported to be dominated by a140 rather than a142 [ 23 , 77 ] . upon 6 months of age ,
furthermore , a recent study showed that senile plaques in cortex and hippocampus can be detected in 3-month - old males but barely in females of the same age which means that a plaques occur earlier in males .
this finding matches our observation that changes of theta and gamma power in ca1 region were firstly observed in males and later in females and stresses the tremendous importance of gender - specific analysis in ad mouse models .
how exactly seizure incidence and power alterations of ca1 theta and gamma are linked with a abundance , a142/a140 ratio , or initial occurrence of plaque formation has to be explored by future studies .
recent studies by in the cortex of appsweps1de9 mice suggest that there is a lack of gabaergic perisomatic synapses of basket cells on the surfaces of cortical pyramidal neurons that are in close contact with amyloid plaques . as perisomatic gabaergic synapses exhibit a dominant influence on the output behavior of pyramidal neurons , their structural impairment may result in hyperactivity of the neurons in close proximity to amyloid plaques .
gamma band rhythmogenesis is known to be inextricably tied to perisomatic inhibition , particularly via gaba a receptors .
as interneurons primarily determine the power of faster oscillations such as gamma [ 8284 ] , it seems unlikely that interneuronal pathology is solely responsible for the observed cortical gamma increase reported by [ 31 , 85 ] . generally , multiple mechanisms contribute to the generation of fast and slow oscillations , for example , cellular resonance properties [ 86 , 87 ] , recurrent excitation , excitation - inhibition interaction [ 89 , 90 ] , negative feedback , gaba receptor kinetics [ 83 , 9294 ] , cellular bursting , or pacemaker activity [ 96 , 97 ] .
thus , hypothesized that multiple factors including basal forebrain projections and local hyperexcitability of cortical pyramidal cells based on increased resting membrane potential may account for the observed alteration in cortical gamma power .
interestingly , reported no changes in any hippocampal eeg range including gamma when recordings were done at 16 - 17 weeks of age .
importantly , the theta and gamma changes observed in our appsweps1de9 study predominantly occur at 18 - 19 weeks of age thus likely to be related to ad progression . multiple hippocampal theta and gamma rhythm generators and underlying ionic mechanisms have been suggested and further studies are necessary to unravel the detailed mechanisms underlying gender , age - specific , and activity - dependent alterations during ad development .
our results demonstrate that hippocampal and cortical seizure and eeg power characteristics are related to both age and disease progression and clearly display gender specificity in the appsweps1de9 mouse model .
these findings further point to the emerging role of novel gender - specific eeg fingerprints as potential early biomarkers of ad in the future . | alzheimer 's disease ( ad ) is a multifactorial disorder leading to progressive memory loss and eventually death . in this study
an appsweps1de9 ad mouse model has been analyzed using implantable video - eeg radiotelemetry to perform long - term eeg recordings from the primary motor cortex m1 and the hippocampal ca1 region in both genders . besides motor activity
, eeg recordings were analyzed for electroencephalographic seizure activity and frequency characteristics using a fast fourier transformation ( fft ) based approach .
automatic seizure detection revealed severe electroencephalographic seizure activity in both m1 and ca1 deflection in appsweps1de9 mice with gender - specific characteristics .
frequency analysis of both surface and deep eeg recordings elicited complex age , gender , and activity dependent alterations in the theta and gamma range .
females displayed an antithetic decrease in theta ( ) and increase in gamma ( ) power at 18 - 19 weeks of age whereas related changes in males occurred earlier at 14 weeks of age . in females ,
theta ( ) and gamma ( ) power alterations predominated in the inactive state suggesting a reduction in atropine - sensitive type ii theta in appsweps1de9 animals .
gender - specific central dysrhythmia and network alterations in appsweps1de9 point to a functional role in behavioral and cognitive deficits and might serve as early biomarkers for ad in the future . |
the increasing adoption of laparoscopic or ( laparoscopic - assisted ) colorectal surgery as standard practice in colorectal cancer management has resulted in reduction in length of stay and improved patient experience .
however , one complication , particular to laparoscopic but not to open surgery , is post - operative subcutaneous emphysema .
although there have been numerous reports of this following laparoscopic upper gastrointestinal and gynaecological surgery , episodes of subcutaneous emphysema following other laparoscopic procedures such as extraperitoneal renal and colorectal surgery are few .
subcutaneous emphysema of the scrotum developing 9 days post endoscopic retrograde cholangiopancreaticography has been reported , though there are no documentations of a significant post - operative delay in manifestation of this complication in any form of laparoscopic surgery . in a retrospective single centre review of 889 laparoscopic resections for colorectal cancer ,
subcutaneous emphysema was the most common intra - operative complication at 1.65% , though there were no reports of subcutaneous emphysema for patients undergoing left hemicolectomy in particular ( most were in anterior resection of the rectum ) and there were no such complications recorded for any laparoscopic colorectal patient in the post - operative period .
in this report , we present the first case of massive subcutaneous emphysema which developed on the third day following laparoscopic left hemicolectomy . to perform the literature review for evidence for this case report , medline and embase were searched on 29 jan 2014 using the key terms : laparoscopy , colorectal cancer , subcutaneous emphysema ; works in all languages were considered .
a 52-year - old female patient , with a large sigmoid tumour and associated mesenteric nodal mass , ( detected following colonoscopy and ct staging after referral from the national bowel screening programme , ) was admitted for an elective laparoscopic left hemicolectomy . of note , she had undergone extensive oral dental surgery ( apicectomy and retrograde filling ) for a recurrent dental pathology 2 months prior to her colorectal surgery .
she was otherwise fit and well with no underlying lung pathology ( asa i ) .
the patient underwent uneventful endotracheal intubation and received standard antibiotic prophylaxis consisting of gentamicin and metronidazole .
she was placed in lithotomy ( slight trendelenburg ) position and 1014 mmhg carbon dioxide ( co2 ) pneumoperitoneum ( flow 20 l min ) was maintained throughout ( for an estimated time of 130 min ) .
the initial end tidal co2 ( etco2 ) at intubation was 4.1 kpa : it was documented as 5.5 kpa at the end of the procedure .
the mean value of documented etco2 during pneumoperitoneum was 5.24 kpa ( sd 0.38 ) .
standard laparoscopic dissection , aided by pneumoperitoneum , of tissue planes was carried out and the specimen was delivered through a self - retaining atraumatic retractor lining the extended midline wound .
the anastomosis was performed laparoscopically using a circular stapled gun and air - leak test was negative .
the patient recovered well post - operatively , with a good level of mobilisation and tolerating oral intake . on the third post - operative day
, she developed surgical emphysema on her chest and neck , confirmed by a chest x - ray which showed a massive pneumoperitoneum and subcutaneous emphysema without definite evidence of pneumothorax .
the only systemic abnormalities were : a non - compromising borderline tachycardia and oxygen saturations between 94 and 95% on air .
further chest x - ray reported no significant interval change except for a suggestion of pneumomediastinum .
a non - tender erythema of her right flank appeared , consistent with cellulitis except for lack of tenderness .
crp rose to 367 ( white cell count remained normal ) and intravenous antibiotics were administered but discontinued after 2 doses , as a senior opinion determined there was no sign of sepsis .
the subcutaneous emphysema resolved spontaneously and the patient was fit for discharge on day 6 . on clinic review ,
all symptoms had resolved and she was referred for adjuvant chemotherapy for a moderately differentiated adenocarcinoma , staged as iii ( t3n2m0 ) , positive for extramural vascular invasion .
clinical manifestations of hypercarbia including subcutaneous emphysema developing during or very shortly after ugi procedures and tep procedures have been well documented .
it has also been postulated that the incidence of surgical emphysema might be as high as 34% , if assessed radiologically via chest x - ray , after pelvic laparoscopic surgery ; mcalister instead stated that up to 56% of the patients who underwent laparoscopic cholecystectomy within the previous 24 h had subcutaneous emphysema on a computed tomography .
hypothesised pathophysiology of subcutaneous emphysema includes tracking of gas along fascial planes from port sites ( increasing number of ports giving rise to increasing incidence of emphysema ) , or through diaphragmatic defects . factors increasing this risk include age of patient ( perhaps due to weaker tissues bonding fascial planes ) , increased number of ports , increased intra - abdominal gas insufflation pressures and long operating times , allowing increased absorption of carbon dioxide .
we are unaware of an instance , prior to this case report , when 72 h have passed before the first signs of subcutaneous emphysema developing after a colorectal procedure .
hypercarbia , although not confirmed by arterial blood gas sampling in our case , may explain some of this patient 's physiological derangement : a resultant acidosis could explain the tachycardia .
a massive pneumoperitoneum may also have caused splinting of the diaphragm and decreased oxygen saturations .
other cases of post - laparoscopic subcutaneous emphysema include an episode presenting after failed ercp in a patient after bariatic surgery , where gas was thought to pass through a weakened intestinal wall into the retroperitoneum ; an exploratory laparotomy for persistent leak excluded a perforation .
risk factors for hypercarbia related injuries include an operating time greater than 200 min , age > 65 and six or more ports ) .
subcutaneous emphysema has been shown to be more common when higher co2 insufflation pressures are used , though in this case a pressure of 10 mmhg was maintained throughout .
possible explanations include a latent pocket of carbon dioxide in the abdominal wall which disseminated only later , after mobilisation of the patient ; a gas leak from the anastomosis , later resealed ; a small iatrogenic mucosal disruption , resulting in retroperitoneal air leak , settled spontaneously . on the other hand , an iatrogenic airway injury or a pneumothorax appears unlikely on account of clinical and radiological findings . in our patient ,
the observations remained stable until the development of subcutaneous emphysema on the third post - operative day . after this time
although arterial blood gases were not deemed necessary , retrospectively this new tachycardia could have been a response to hypercarbia , following delayed absorption of carbon dioxide .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal .
aj , up and se performed a search of the literature and wrote the report , under supervision of ajw . | highlightswe encountered a peculiar case of surgical emphysema 3 days after an uneventful laparoscopic left colectomy.we reviewed the literature to ascertain the presence of similar episodes.albeit unable to determine the cause of the event , this is the first case of delayed emphysema after a laparoscopic colorectal procedure . |
ectopic pregnancy ( ep ) is a term used for implantation of blastocyst outside the natural cavity of uterus ( 1 , 2 ) .
the prevalence of ep has been doubled since 1960 and comprises 2% of pregnancies diagnosed at the first trimester ( 3 ) . in the developing countries like iran ,
10% of women diagnosed with ep , can not survive , as they refer to the hospital very late ( 4 ) .
the prevalence of ep has increased owning to high occurrence of prior damage to the fallopian tubes , due to the abdominal operation ( 5 ) , infertility ( 6 ) , using artificial reproductive technology ( 7 ) , infections like chlamydia trachomatis ( 8) , and using contraception devices , which contains only progesterone ( 9 ) .
considering high prevalence of ep , finding efficient and safe methods of treatment has gained top priority .
if the patient s hemodynamic condition is stable , with no significant symptoms , and low free intraperitoneal fluid detected on ultrasound ( 10 ) , medical treatment is preferable in which the single dose of methotrexate ( mtx ) is prescribed in 34% - 69% of cases ( 11 ) .
however , when the fallopian tubes remained unruptured , the medical treatment is preferable for preserving fertility ( 12 ) . also , medical treatment is more feasible , affordable , and accepted by patients .
but it is not clear that which medical protocol ( single , double , or multiple doses ) is preferable .
however , the success of multidose therapy is more than single dose in most of the studies ( 13 ) .
some studies have found that double dose is preferable due to its few side effects ( 14 ) .
there are few studies on comparing the effectiveness of single dose with double dose administration of mtx , therefore this study was designed to compare the effectiveness of single and double dose of mtx in treatment of ep .
this research is a randomized controlled trial study conducted on 76 patients with ectopic pregnancy referring to imam khomeini hospital of ahvaz city , iran during 2012 - 2013 .
the design of this study was approved by the ethics committee of ahvaz jundishapur university of medical sciences , ahvaz , iran ( reference number : ajums .
imam khomeini hospital is specialty teaching hospital affiliated to the ahvaz jundishapur university of medical sciences and is the referral hospital in the khuzestan province .
inclusion criteria were as follows : healthy women with stable hemodynamic condition and -hcg value of less than 15000 miu / ml .
women who had a history of liver and kidney disease , blood dyscrasia or breastfed were excluded from the study ( 14 ) .
patients were randomly assigned in two groups of single and double dose by the method of enclosed envelope include block randomization ( with ratio 1:1 ) . according to similar studies , the lowest success rate for single dose and double dose
have been reported 55% and 77% , respectively ( 14 ) . considering 95% confidence interval the sample size was calculated as 76 patients ( 38 in each single or double dose group ) according to the following equation : where = 0.05 and = 2% . in the single dose group , 50 mg / m mtx was administered at day 0 , while in the double dose group ; patients received two doses of 50 mg / m at days 0 and 4 .
the values of -hcg in both groups were measured on the fourth and seventh day of hospitalization .
about 15% decrease in -hcg level was considered as a successful treatment . in case of treatment s failure , elective surgery or administration of extra dose of mtx
patients remained hospitalized during the intervention period and were checked for the level of -hcg every 48 hours . before prescribing mtx , blood tests , kidney and liver transaminase levels , creatinine and urea levels were measured and if the patients showed any problem , they were excluded from the study . during the intervention , the following signs were compared between two groups and considered as drug complications ; if liver transaminase increased 1.5 times more than normal , or creatinine increased to 1.5 mg / dl , wbc count decreased to 2000 , or platelets declined fewer than 100,000 ( 11 ) . in order to compare the effectiveness of mtx doses in different -hcg levels or mass sizes
; patients were classified in 3 groups of lower than 2000 miu / ml , 2000 - 4000 miu / ml , and more than 4000 miu / ml based on -hcg levels and in 3 groups of lower than 2 cm , 2 - 4 cm and more than 4 cm based on mass size .
every woman remained hospitalized under the precise observation until their -hcg value reached 200 miu / ml .
the side effects of mtx , as well as function of liver and kidney were checked in each participant one week after completion of treatment and patients were asked for complications such as nausea , vomiting , and gastrointestinal symptoms .
the chi - square , independent t test , and logistic regression were used for analyzing data .
the normality of data was checked using kolmogorov - smirnov test and as a result , all continuous variables were normal .
in the single dose group , 50 mg / m mtx was administered at day 0 , while in the double dose group ; patients received two doses of 50 mg / m at days 0 and 4 .
the values of -hcg in both groups were measured on the fourth and seventh day of hospitalization .
about 15% decrease in -hcg level was considered as a successful treatment . in case of treatment s failure , elective surgery or administration of extra dose of mtx
patients remained hospitalized during the intervention period and were checked for the level of -hcg every 48 hours .
before prescribing mtx , blood tests , kidney and liver transaminase levels , creatinine and urea levels were measured and if the patients showed any problem , they were excluded from the study . during the intervention , the following signs were compared between two groups and considered as drug complications ; if liver transaminase increased 1.5 times more than normal , or creatinine increased to 1.5 mg / dl , wbc count decreased to 2000 , or platelets declined fewer than 100,000 ( 11 ) . in order to compare the effectiveness of mtx doses in different -hcg levels or mass sizes ; patients were classified in 3 groups of lower than 2000 miu / ml , 2000 - 4000 miu / ml , and more than 4000 miu / ml based on -hcg levels and in 3 groups of lower than 2 cm , 2 - 4 cm and more than 4 cm based on mass size .
every woman remained hospitalized under the precise observation until their -hcg value reached 200 miu / ml .
the side effects of mtx , as well as function of liver and kidney were checked in each participant one week after completion of treatment and patients were asked for complications such as nausea , vomiting , and gastrointestinal symptoms .
the chi - square , independent t test , and logistic regression were used for analyzing data .
the normality of data was checked using kolmogorov - smirnov test and as a result , all continuous variables were normal .
sociodemographic characteristics of the patients such as age , parity , and history of abortion are listed in table 1 .
as it is evident in table 1 , demographic characteristics and the mean of -hcg level in two groups did not show any significant difference .
as demonstrated in table 2 , the success rate of single dose was 69% and in double dose group , it was 79%
the rate of operation was 19% and 16% in single and double dose groups , respectively ( p = 0.76 ) .
the necessity of extra dose of mtx in the single dose grope was 14% and in the double dose group , it was 6% ( p = 0.23 ) .
the two therapeutic methods were compared with regard to 3 different levels of -hcg and mass size .
the results of logistic regression showed that there were no any significant differences between two groups in the rate of success considering the level of -hcg and mass size ( p > 0.05 , table 3 ) .
results showed that only the rate of operation at the -hcg level of 2000 - 4000 miu / ml was lower in the double dose compared to the single dose group ( p = 0.05 ) .
duration of hospitalization in double dose group was significantly lower than that of the single dose group ( 11.55 vs. 14.76 d , p < 0.001 ) .
the rate of complications in double dose group was more than single dose group , but no significant difference was observed ( p = 0.32 ) .
complications included vomiting and gastrointestinal symptoms and no blood or urine disorders were reported after prescription of mtx among patients .
this study designed to compare the effectiveness of single and double doses of mtx in patients with ep .
our research showed that double dose method might induce better response with regard to necessity of operation and extra doses of mtx compared to single dose method .
therapeutic success of double dose method in our research was 79% , which was lower than hamed et al .
study result ( 82% and 88% in the single and double dose , respectively ) ( 13 ) .
this discrepancy may be due to the level of -hcg in our patients that was more than that of the hamed et al .
they also did not notice any significant relation between therapeutic method and response to the treatment in their research . in a study by barnhart et al . in which they administered 50 mg / m mtx on days 0 and 4 , results showed that the success rate was 87% and the fallopian tubes ruptured in 3% of patients .
the reported side effects were mild and transient and most women were satisfied with the treatment ( 14 ) .
the rate of success in our study in the double dose group was less than that observed by barnhart et al .
study ( 14 ) that may be due the overall level of -hcg in their study which was lower than that of our study .
( 15 ) in a study on 34 patients who received single dose of mtx , noticed the higher rate of success in the group with -hcg level lower than 2000 miu / ml compared to the group with -hcg level more than 4000 miu / ml ( 78% versus 33.33% ) .
they concluded that the mean of -hcg is an important factor in response to treatment ( 15 ) . in our study , the rate of success was also higher in the group with -hcg level lower than 2000 miu / ml ( 91.7% and 77% , respectively ) and the mean of -hcg was also lower in patients responded to the treatment than cases who did not . in a study by alleyassin et al .
, they compared the effect of single dose with multiple doses of mtx in selected women .
the results showed that the rate of success in the single dose was 88.9% , while in the multiple doses , it was 92.6% ( p > 0.05 ) .
the researchers concluded that single dose can be considered as the first choice of treatment in selected patients ( 16 ) .
the higher level of initial -hcg in the single dose group in our study may be a reason for this dissimilarity ( 3249 1427 vs. 3146.9 2389 ) . according to our findings , the single dose of methotrexate ( compared to the double dose ) is effective in cases of ep with primary low level of -hcg .
however in cases with higher level of -hcg or probability of tube rupture , administration of double dose is recommended .
also the duration of hospitalization in the double dose group was significantly lower compared to this time in the single dose group .
this is the first time in iran that we compared the single dose versus double dose of mtx for treatment of ectopic pregnancy .
results of this study have been considered as a part of treatment regimens in the imam khomeini hospital after completion of this study . | background : ectopic pregnancy ( ep ) is one of the most dangerous complications of pregnancy and without prompt diagnosis and treatment , it could become a major cause of maternal morbidity and mortality.objectives:in this randomized controlled study , we compared single and double dose of methotrexate ( mtx ) therapy in the treatment of ectopic pregnancy.patients and methods : this study was performed on 76 patients who were admitted to obstetrics ward with primary diagnosis of ectopic pregnancy based on their medical history , physical examination , beta subunit ( -hcg ) level , and transvaginal ultrasonography . using random block allocation ,
the patients were classified in two groups of single dose and double dose administration of mtx . in single dose group , 50 mg / m2 of mtx was given at day 0 and in double dose group , the patients received two doses of mtx at day 0 and 4 .
the level of -hcg was measured at day 0 , 4 , 7 in both groups .
the successful treatment was defined as 15% reduction in -hcg level between day 4 and 7 .
the two groups were compared with each other with regard to their need for operation , or extra dose of mtx ; duration of hospitalization ; and mtx complications.results:results showed that the rate of success in double dose method was more than single dose one ( 79% versus 69% ) but the difference was not significant ( p = 0.29 ) .
although the need for operation and extra dose of mtx were lower in the double dose group ( 15.8% vs. 18.8% and 5.26% vs. 13.2% , respectively ) , these differences were not significant too .
duration of hospitalization was significantly lower in double dose compared to the single dose ( 11.55 d vs. 14.76 d , p < 0.001).conclusions : single dose therapy of mtx has sufficient power and efficacy in the treatment of ectopic pregnancy , however in patients with higher serum level of -hcg , the successful treatment increases by using double dose method . using double dose
also could decrease the necessity of operation , re - administration of mtx , and duration of hospitalization . |
research in personalized medicine seeks to achieve optimal clinical outcomes through the use of innovative biomarker discoveries to develop drugs that best suit a specific group of patients . to derive best - fit treatment options for a specific patient group ,
various signaling pathways are thoroughly analyzed to identify altered molecular circuitry that initiates and maintains the clinical phenotype of the disease . for cancer , this altered signaling promotes a cascade of molecular events , that is , cell- or tissue type - dependent , and this relationship gives rise to a specific biomarker set that has a direct association with the cancer phenotype .
the unique genetic profile of an individual patient 's cancer will generate specific gene expression signatures and modifications of genes / mirna , proteins , and metabolites .
current and future technologies will produce a flood of data , not only from the laboratory bench but also from clinical sources , and the association of these data with a specific cancer phenotype is expected to be more sensitive and to have a higher specificity that will allow increasingly accurate clinical decision making . mutations are often associated with specific cancer malignancies , and the combination of well - documented mutations with better - fit drugs has the potential to provide a good clinical outcome with fewer side effects for patients . for example , cyp2d6 genotyping at the time of breast cancer diagnosis provides prognostic information regarding the effectiveness of tamoxifen treatment . if a poor clinical outcome is predicted , then additional data and evidence - driven clinical decisions may allow better alternative therapy options for those patients . with recent advances ,
the er , pr , and her2 status of breast cancer patients can be used to make therapeutic choices that include a customized tamoxifen regimen to prevent disease recurrence .
the next generation of sequencing and -omics technologies will continue to improve our ability to recognize cancers , improve treatments , and track the health of survivors .
-omics now represent many pathways and molecular signatures that are directly involved in triggering cancers , and these research data can be applied to diagnosis , prognosis , and treatment decisions . as the field of personalized medicine evolves , scientific and clinical data are expected to converge in a common central database , which will facilitate the exchange of data that can be transformed into valuable information that will aid clinical decisions , enable customized treatments , and generate new advances for cancer medicine .
these developments would be cost effective for both patients and health care practitioners because a proper course of action could be identified using the stored data , which will help avoid the use of unnecessary tests and ineffective therapies . with the implementation of the affordable care act
obamacare in 2014 , economical assays that provide accurate results will be needed to determine the most effective treatments and ensure the best outcomes . in evaluating the cost of screening , assay costs and the cost effectiveness of drugs
must be considered and should correlate with the cost of biomarker detection in patient samples . screening assays will provide tools for making the most accurate clinical decisions , such that the customized treatment represents a best fit , if not a match , and targets molecular changes that occur in the patient 's tumor .
companion assays for each targeted drug will maximize the treatment benefit options for a specific cancer . minimally invasive procedures for obtaining small biospecimens from patients will also help drive down medical costs to satisfy the new regulations . the combined scientific and clinical information from each cancer patient can be further analyzed and compared precisely with traditional treatments , and any new treatments with companion diagnostics can be tested in clinical trials .
thus , elucidating the molecular profile of each patient involves testing for robust biomarkers with high sensitivity and specificity for particular cancer phenotypes and requires high - quality biospecimens and well - organized tissue banking .
scientific information must be integrated with clinical information derived from well - documented electronic medical records ( emrs ) using informatics tools , although to date the importance of this integration has been largely neglected by the research and clinical community . in combination ,
tissue banking , bioinformatics , and emrs are front - end requirements for enhancing personalized medicine , and they represent key resources that will determine the path of progress in translational research to support clinical applications .
bioinformatics encompasses a combination of statistics , molecular biology , and computer science to store and analyze biological data , while tissue banking involves procurement of tissue or tumor samples during medical procedures and their collection and storage in a tissue repository . by integrating patient information and experimental results , novel biomarkers
can be discovered that may have a major effect on the way patients receive care and could also greatly influence drug and diagnostic customization .
biomarkers can be proteins , genes , metabolites , or even methylation patterns that are used to detect genetic tendencies for specific types of cancers and other diseases ( table 1 ) .
depending on the cancer , certain diagnostic tests are performed , and if a given biomarker is found in a patient sample , it will guide the choice of treatment . in the case of melanoma ,
for example , a braf mutation test is used to determine if a braf v600e mutation exists in the patient sample , and if present , that patient will receive a customized treatment with the drug zelboraf , which is effective only for brafv600e - positive melanomas .
nonsmall cell lung cancer ( nsclc ) patients carrying an alk mutation are treated with the drug xalkori , which is specific for this type of mutation , whereas nsclc patients having an egfr mutation would receive alternative customized treatments .
these well - studied biomarkers enhance the ability of clinicians to determine a course of treatment with minimal toxicity .
however , there remains a need for technologies to assist with the discovery and transformation of biomarkers to diagnostic uses .
one approach is the use of aptamers , small dna / rna oligonucleotides having high selectivity for their target , which can help avoid the limitations imposed by antibodies in diagnostic applications . to initially identify potential cancer - specific biomarkers ,
biomarker discovery applications require bioinformatics data , information from which can then be used for analysis of samples stored in tissue banks .
sample quality and the associated clinical information are also important factors in biomarker discovery because data from all components are required to complete this process [ 9 , 10 ] .
table 1 includes a list of currently available drugs in cancer therapy , developed after identifying relevant biomarkers and companion diagnostics [ 1141 ] .
given the variety of data and samples being collected and advances in personalized medicine , the importance of collaborations between scientists and clinicians , as well as industry and academia , becomes evident .
-omics technologies , and even issues with biomarker - related trials can be approached only through collaboration .
there are scientific uncertainties present in all areas under study , which medical research alone can not address .
however , through a transdisciplinary scientific methodology , these disparate areas can be combined to offer innovative and unique diagnostic and treatment plans .
in the past two decades , efforts to create and maintain tissue banks have provided a foundation for future research toward personalized medicine in cancer . after potential biomarkers have been identified through the use of bioinformatics and
an experimental design has been put into place , tissue banks come into play . if , for example , an institution wants to examine biomarkers associated with ovarian cancer , a query of the records of the tissue bank will return sample catalog numbers pertaining to ovarian cancer enabling experiments to commence .
the most important and potentially problematic aspect of a successful tissue bank is the quality of stored samples .
proper procurement methods , sample handling , and the bank itself are crucial since biomarker investigations performed using low - quality samples will likely generate erroneous and misleading data . additionally , relatively few patients consent to have their biosamples banked , possibly due to a lack of understanding regarding the use of their tissue and the concern that the information gained could be used against them in the future .
therefore , to improve the rate of consent , improved education of the public and patient populations regarding the safety and value of such donations is needed .
in addition , the consent process , which involves long , difficult to understand forms that must be read and understood , could be made less burdensome .
one approach to improve education would be to employ models for software engagement that have been successful in commercial and educational settings .
these include 3d immersive settings that emulate real - world situations , such as in the second life virtual world platform , where dynamic choices and outcomes can result in good or bad medical outcomes .
some features of multiuser games would likely be of sufficient interest to sustain the attention of a variety of potential donors and increase patient awareness of tissue donation .
constructing and deploying these sorts of educational tools in public and medical venues , such as in malls and doctors ' offices , or in free apps for smart phones or tablets could be of value in informing patients of the importance of tissue banking as applied to research discoveries and treatment successes . to increase the number and variety of biosamples
one promising approach for increasing the rates of consent to donate biospecimens was inspired by modern online software that encourages interactive learning for casual users .
such game - like interfaces were shown to be useful in medical education and training .
better - informed patients who consent more often will enrich the number and variety of samples in the tissue banks , which will support the data analysis and integrated information bases that can lead to discoveries and treatments .
an example session might involve an individual interacting with a public kiosk that allows some user input and provides an immersive environment where the user engages another character who plays the role of a doctor asking for consent to use a tissue sample for research purposes . during the game - like session , the user could be led through a set of choices based on their actions and responses in realistic scenarios . the encounters and selection sequence could be engineered such that consent form issues are clarified .
complex issues such as biomolecular interactions and genetic conditions could be illustrated in attractive 3d renderings , so as to better visualize biological systems and to focus more precisely on why certain therapies are suggested . during the session ,
the importance of consenting to donate biospecimens could be reinforced and the user rewarded by illustrations of improved health of other patients based on more complete information gleaned in part from donated tissue samples .
the play could also educate the potential donor in how such general consents could eventually be of personal benefit in the event of negative health conditions later in their life . while tissue banks archive and
provide biospecimens for translational research and healthcare innovations , there are challenges to be faced before sample procurement from donors .
obtaining informed donor consent is a major consideration , as the health care system considers informed consent to be legally and ethically required to ensure donor data security . moreover ,
informed consent is an autonomous act , and by signing the consent , the patient / donor confirms that he / she understands the risks and benefits of research , is protected from exploitation , and learns the purpose of the research for which the donations are made .
recent research by beskow et al . suggests that combining a simplified consent form with supplemental materials aimed at patients who wish to have additional information about the research would minimize the complications in understanding research goals .
similarly , there are opt - in and opt - out criteria for participating in specified research .
opt - out methods are passive allowing the participants / donors to decide whether to be excluded from the research .
often , the opt - out approach provides little information to the donor regarding the exact research goals .
opt - out also provides some choices for participants / donors within a limited timeframe , which helps to procure more samples to accelerate research .
the opt - in method signals the willingness of participants / donors to take a more active role in the research ; this method receives more public acceptance with more information and participant education .
the broad consent given by a participant permits use of their biospecimens and personal medical information for future unspecified research under appropriate irb regulations .
criterion , which allows the donor to be informed about the various processes involved in tissue bank functions but with limited or no information about the nature of future research .
broad consent has advantages over specified informed consent because it allows biospecimens to be used for diverse research activities and minimizes the practical difficulties in transporting consent to the context of tissue banks .
patients are more likely to give consent if they have been educated with respect to the benefits of tissue banking prior to the hospital visit ; thus , public awareness plays a vital role in encouraging tissue donation .
this can be accomplished by individuals that represent patients / donors and their families , who have already consented and donated biospecimens .
these alumni groups or individuals can speak in family gatherings or community events and clarify the confusion and fear that may accompany the cancer diagnosis and anticipated medical procedures . in the absence of reliable , accurate information , misconceptions about additional procedures , treatments , and tests can deter a patient from making a well - informed decision .
conversations without the confusing medical jargon with friends and community members who have gone through the experience can help potential patients / donors and their families understand the importance of donation for tissue banking . a small , friendly conversation with the person next door
atmosphere can make it personal enough for the patient / donor to give consent for tissue banking .
this type of personal conversation can be adopted for a national stage at patient advocate conferences or convention .
the fact that a specific medical condition becomes a top search query on the internet on the same day it is described by the national media illustrates the eagerness of general public and communities to learn when information is distributed appropriately .
we expect similar responses as previously mentioned when efforts are provided to the general public and patient communities regarding the importance of tissue banking and educate about the role of tissue banking for innovations related to personalized medicine .
biospecimen information and corresponding molecular and clinicopathological data is currently stored primarily using xml for web - based exchange of information .
this system consists of different servers including web , application , database , authentication , and authorization .
the system connects with other relevant databases within a hospital enabling storage and retrieval of clinical , pathological , and personal data with highly secure access . ensuring security for accessing records is provided via digital codes or smart cards for users .
software interfaces play a role in storage and retrieval of larger data sets associated with biospecimens detailing storage location , history , and storage time providing easy traceability of samples .
the laboratory information management system ( lims ) is a flexible , expandable , and secure software interface used for storing and retrieving large amounts of data generated through the processing of biospecimens in research labs .
for example , screenings for genetic mutations in an automated environment require data storage , retrieval , and tracking .
lims helps minimize human errors in data processing by communicating with lab equipment , robots , and databases and enables easy retrieval of data .
the implementation of bims ( biobank information management system ) ensures the integration of data from different sources , such as various research institutions , which may employ different formats and procedures .
extraction , de - identification , consolidation , abstraction , and query are individual components that handle and manage data .
the extraction component receives data from external sources , which are continuously updated and/or are in different formats depending on the system used to generate the data ( e.g. , text files , spread sheets ) .
deidentification attaches a unique identifier to each data item stored in bims and separates personally identifiable information from clinical and scientific data .
abstraction supports data presentation and control when researchers / users access the data through queries .
the web - based query interface allows access for retrieval of data from the database .
figure 2 illustrates an integrated knowledge environment through which personalized medicine can be approached . within this environment
, clinicians can determine if a patient has cancer and what course of treatment should be taken .
biomarker analysis will provide the information necessary to determine whether the patient should receive a conventional or customized treatment plan .
for example , as noted earlier , the small fraction of nsclc patients carrying a chromosome rearrangement resulting from an alk mutation will have a poor outcome with conventional therapy but will do well with a personalized treatment that includes xalkori , which is ineffective in a majority of nscls patients .
all experiments are dependent on the procurement and availability of high - quality , viable tissue specimens ; thus , from the first step of procurement , it is crucial for the sample to be properly handled .
collection , storage , and distribution of high quality tissue and blood samples require information supplied by bioinformatics data in concert with a reliable emr system to link clinical data of interest .
a significant key to proper tissue banking involves proper acquisition of samples by pathologists , who are responsible for determining what should be stored in the tissue bank by identifying the sample nature and origin , which makes pathology central to communication between scientists and clinicians .
as in bioinformatics , tissue banking requires collaborative efforts between academia , health institutions , and pharmaceutical companies .
there are many issues , however , that pertain to tissue banking , including the loss of samples due to traceability and documentation errors and the need for coordination between multiple disciplines during the entire procedure . the cancer genome atlas project ( an nci initiative ) assessed the quality of samples acquired from dozens of tissue banks and produced the surprising result that only one percent of the samples assessed were viable .
in addition , most of the tissue banks that supplied these samples had no proper catalog of samples that were stored in their facilities [ 55 , 56 ] .
well - established labs that have implemented a precise bank setup can maximize their research potential and , eventually , contribute to affordable health care diagnostics for patients as well as clinicians .
biobanks such as the uk biobank , biobanking and biomolecular resources research infrastructure ( bbmri ) , and biobank japan are among those that have created networks of organizations that manage millions of patient samples .
stored samples can be used to analyze cancers at various stages down to a molecular level .
because of the availability of samples from these biobanks , even for rare cancers , there would be no delay for sample collection since samples would already be available for experimentation .
these samples can be used for mechanistic studies for investigating the role of biomarker in disease progression or used with clinical data points and records ( emrs ) to predict treatment options , drug responses , and susceptibility [ 58 , 59 ] . by analyzing tumor samples and applying a comprehensive approach to determining the proper therapeutic regimen
however , if a given sample lacks relevant clinical data from the donor , experimental results will be more difficult to translate into real - practice medicine .
samples available from tissue banks are used for experiments to discover and validate biomarkers to assist in translational research , identify diagnostic and prognostic targets to maximize patient benefit from customized care , and contribute to future diagnosis and treatment options .
the discovery , validation , and implementation phases of research on biomarkers require the resources found in tissue banks .
completion of the process and confirmation of biomarkers will lead to low - cost , yet efficient , and reliable medical care that will result in better patient care .
emrs are used to improve patient care , and to date , their utilization has established high - quality practice - based datasets that are well suited for scientific research [ 61 , 62 ] . a universal , user friendly , google - like electronic medical record ( emr ) system that allows crosstalk between various infrastructures is needed , since novel biomarker discovery is not possible unless clinical data are linked with patient samples that can be associated with clinical outcome . to support this type of initiatives at national level , nci has developed an electronicprogram providing a collection of cancer - related information , known as cabig , and made it available to several hospitals and institutions .
the sole purpose of the cabig program is to assemble emr files digitally in order for clinicians and scientists to facilitate translational medicine .
however , cabig function requires that an emr setup be in place , and since many institutions have not yet instituted a proper emr system , cabig implementation has been slow .
early adopters of emr technologies include sweden and denmark in the eu and kaiser permanente and the veterans health administration in the usa
. even at early stage of emr development , the success of it implementations by these organizations is shown in their increased effectiveness in managing clinical data in electronic format . however , failed emr projects in numerous organizations indicate that the currently implemented system needs to be revised .
the major issues related to it implementations are the complexity of managing the infrastructure , maintenance of alignment between organizations with different governance , managing funding , ensuring involvement of it staff and managers , engagement of vendors , and adapting to changes to improve working methods . in the usa , development of emr
will permit integration of biological data , clinical information , patient information , and clinical outcomes .
large populations or specific groups of patients with selected characteristics could be easily identified with the availability of electronic medical records .
emr can provide a larger number of participants , a wide range of information , and lower research costs . in genomic research ,
emrs facilitate analysis of genetic and molecular information from large subject populations allowing studies to be more powerful than smaller cohort studies . even though there are difficulties associated with using information provided from emrs , the lower cost of research and faster pace of advancement in clinical care help in overcoming the difficulties .
in the usa , larger emr databases are being linked to biospecimens procured from patients / donors covering a wide range of diseases .
analysis of biospecimens resulting in identification of genetic variants or single nucleotide polymorphisms ( snps ) can be correlated and linked with available ehr information regarding parameters such as smoking history , obesity , cardio vascular diseases , and hypothyroidism . a recent study using a descriptive , qualitative approach for exploring the experiences of primary healthcare providers who use ehrs in their ongoing practice identified factors that support and hinder the use of ehrs in healthcare .
factors that support the use of ehrs include improvement in efficiency of patient care and confidence in it systems and software , whereas factors that hinder the use of ehrs include it challenges related to computer usage such as scanning , electronic connectivity , and attaining proficiency in computer use .
generally , these factors that hinder use of emrs can be addressed through training to enhance computer skills , along with consistent use of emrs and data entry . to apply innovative approaches in science and to produce the vast amounts of data required for biomarker analysis , it is essential to incorporate clinical data , which can only be made available through emrs . translating scientific discoveries into medical practice is the most challenging part of personalized medicine , but once the scientific information ( i.e. , biomarkers , mutations , pathways , and drugs ) is integrated with clinical data ( i.e. , survival , relapse , pathology , medications / treatments , and response ) , the translational power of bioinformatics will be apparent .
other emr advantages include communication between clinicians , scientists , and other health care providers , as well as notification of health care practitioners of common chemotherapy - related errors . however , there are certain conflicts regarding the ethical issues related to emrs . exposure of health records by mistake , lack of security of system , and stealing can breach the confidentiality and fidelity of emrs which might affect the treatment of patients [ 68 , 69 ] .
emrs require great attention to the challenges of electronically documenting care , the cost of building a large - scale emr system to replace paper notes , and the cost of training employees .
when a universal emr is in place , it will require management in structured data security , data recovery , and infrastructure inspection , which will be a general requirement for all medical institutions .
insufficient medical information technology ( it ) infrastructure and a lack of digitized clinical data are other issues that will almost certainly arise .
cancer care is complex , and to facilitate management of oncology patient records and workflow , detail - intensive data need to be organized in emrs .
many cancer therapies involve substantial patient illness , and emrs are an important clinical tool to improve patient safety , suitability , and efficiency , as well as patient - centeredness .
bioinformatics can enable clinicians to answer a fundamental question : based on all that is known about an individual patient , including disease characteristics , lab results , genomic , proteomic , and metabolomic information , what are the similarities to other patients who had good outcomes , and what is the best therapy for this patient with this disease ?
the progression of biomarker discovery is impossible without bioinformatics , which connects individual discovery processes , including experimental design , study execution , and bioanalytic analysis .
bioinformatics has dramatically progressed in the past decade with more than one million published articles in oncology research available in pubmed . in the sphere of bioinformatics , large amounts of pertinent
-omics data and information from the human genome are available for oncologic approaches to develop biomarker - related therapies .
the nci bioinformatics infrastructure cabig makes available information from multiple studies to support new scientific efforts .
the infrastructure also contains details of experiments , protocols , samples used , and specific results , which can be easily searched , compared , and downloaded from the database .
nci 's early detection research network ( edrn ) developed this program so that cancer institutions would have the bioinformatics data needed to support their research .
the edrn exists today because of improvements in oncology research in the past decade that have derived from progress in bioinformatics .
the direction from which studies are approached has been revolutionized by the essential usefulness of bioinformatic analysis of large - scale experimental datasets .
nonetheless , many cancer researchers are not accustomed to using bioinformatics and have not incorporated these advantages into their research
. this will likely change as researchers grasp more fully the significance of the overlap between many areas of cancer research and bioinformatics with respect to data analysis and interpretation .
translational research has been supported by bioinformatics , which has provided critical tools for transforming data into medical practice and has prompted biomarker breakthroughs and drug development ( figure 3 ) .
the use of clinically validated biomarkers will also permit development of cheaper , less invasive tests that will benefit both clinicians and patients .
however , the entire process will fail without the availability of properly curated patient clinical information with which to correlate the biomarker data .
bioinformatic results can not be interpreted in the absence of clinical data and patient history , including treatment , physician 's notes , pathology reports , and signs and symptoms .
the failure to implement use of emrs is a critical aspect that delays integration of patient records with experimental results , yet many healthcare facilities still rely on paper notes .
often , hospital personnel , including clinicians , lack the training , interest , or time to learn and understand the benefits of bioinformatics .
there is no mystery to this , health care providers are occupied with patient care , and there is no reimbursement for entering clinical data into a database .
nonetheless , the participation of clinical personnel in bioinformatics research is vital and must be encouraged .
patient emr information , including demographics and disease characteristics , combined with tissue bank - derived experimental results , can be used to create a personalized patient genomic profile or
information object that can allow clinicians to query integrated bioinformatics systems to compare each individual patient with current and historical information .
intelligent picture of the therapies and treatments that have the highest probabilities for the best outcomes . systems to support clinicians must be fast , easy to access , and must provide clear answers to personalized medicine questions . to identify optimal therapies for each unique circumstance , clinicians need easy access to intelligence
, which is provided through the integration and correlation of critical information stored in disparate and constantly changing data sources .
successful integration requires a three - way alignment of information stored in the patient 's emr , tissue bank pathology test results , and genomic information .
clinical data on patients , diseases , therapies , and outcomes are stored in hospital patient databases and public sources such as nci 's tcga and target databases . to advance the cause of personalized medicine
, these clinical databases should allow easy access to outcomes data for each patient cohort for specific diseases and associated therapies , which , along with related drug safety and toxicity information ( fda drug labels , drugbank ) , is critical to decision making regarding the care of each patient ( figure 1 ) .
kaplan - meier chart information can also allow clinicians to quickly and easily review details , sources , and specifics of evolving information to gain the confidence required to decide on a specific treatment regimen .
bioinformatic support for clinicians should integrate the ability to produce kaplan - meier charts for easy reference . the development of bioinformatics to support personalized medicine has been underway in government , academic , and private hospital research cancer centers for years .
progress is being made toward resolving some of the more complex obstacles for translating and integrating disparate data sources .
although the current state of bioinformatics is imperfect , collaborations between government , academic , and biotech and pharmaceutical companies have produced advances in standards , ontologies , and vocabularies that support integration of critical information . currently available flexible data models provide scientific , medical , and semantic relationships between critical data elements that are often stored in various systems and databases . the virtual integration provided by data models
enables querying , mining , searching , retrieving , and accessing critical information necessary to support accurate decision making .
hl7 standards enable translation and communication of patient information stored in emrs , while gene ontology and nci 's thesaurus help align vocabularies and terms across research databases .
clinical and biological databases such as those hosted by the ncbi as well as others such as reactome and sanger 's cosmic mutation database provide evolving and improving reference databases from which information can be mined and aligned with laboratory data , sequence information , and genetic mutations to help clinicians decide on treatment options with the highest likelihood of positive outcomes .
the synthesis and analysis of information from these various sources can expose relationships between critical data elements that provide intelligence and can identify the best therapeutic course to deliver the best outcome .
academic cancer treatment hospitals ( e.g. , md anderson , mayo clinic ) have led the way in developing software and technologies that allow data integration and emr mining .
commercial software companies are delivering innovative knowledge environments such as sophic 's scan - mark explorer cancer biomarker database , research , mining , and discovery system , which is built on biomax bioxm technology . with software like bioxm
, experimental designs can focus on predictive biomarkers that indicate whether a customized treatment plan is needed for certain patients . in biomarker research ,
scientists examine how biomarkers are formed , how they function , and how that function relates to patient data . before
biomarkers can be adopted for practical use in the format of assays and for detection by various biosensors , patient information and bioinformatics need to be combined to generate hypotheses that can be tested using clinical samples stored in tissue banks .
bioinformatics , in particular , provides an irreplaceable infrastructure to support accompanying multidisciplinary education and research . in the future
, bioinformatics will enable the customization of medical care to the specific genome of each patient rather than providing a single , conventional treatment .
there will be an abundance of information that can only be approached through bioinformatic analysis .
cancer is complicated , and biomarkers promise advancement of early diagnosis and targeted therapies , which would not be possible without bioinformatics tools . in the case of hodgkin 's lymphoma , up to 20% of patients will relapse after receiving conventional frontline therapy . without bioinformatic data analysis to design clinical studies and query
-omics information , these fingerprints of cancer would be difficult or impossible to find .
the strategy that should be adopted is to take potential biomarkers and use them to determine the diagnosis , prognosis , and course of therapy for specific cancers .
biomarker discoveries have the capability of improving early diagnosis , as well as guiding customized therapies and enhanced monitoring after treatment .
thus , the effective use of bioinformatics in clinical setting by analyzing clinical data from emr along with biomarker - derived diagnostic or prognostic assays can provide clinical decision making steps that could be of long - term benefit to patients .
health care practitioners need more training in the areas of bioinformatics and tissue banking , and emr systems need to be in place to provide the necessary components for biomarker discovery that can lead to customized medical care for cancer patients .
training can be accomplished by ( i ) presenting bioinformatics as a helpful toolkit ; ( ii ) promoting improved training of individuals in proper protocols for tissue procurement and storage ; ( iii ) encouraging documentation of all patient information using emrs instead of paper charts ; and ( iv ) fostering further collaborative efforts between clinicians and scientists . methods for providing more information about the benefits of tissue sample donations and increasing informed consent can be improved to ensure richer data sources for determining patterns and personal differences . with these enhancements in place ,
the focus can be shifted to the discovery of predictive , diagnostic , and prognostic biomarkers that will allow proper diagnosis of specific cancers , increase information about a particular disease , and indicate the direction treatment should take .
community - wide efforts for collecting patient information , creation of well - controlled tissue banks , and development of new genomic and proteomic technologies will provide clinicians with the building blocks for achieving personalized medicine and improving patient treatment outcomes
. these efforts will also provide patients with reduced medical care costs . on the long run , adoption and integration of bioinformatic tools into routine clinical use | personalized medicine promises patient - tailored treatments that enhance patient care and decrease overall treatment costs by focusing on genetics and -omics data obtained from patient biospecimens and records to guide therapy choices that generate good clinical outcomes .
the approach relies on diagnostic and prognostic use of novel biomarkers discovered through combinations of tissue banking , bioinformatics , and electronic medical records ( emrs ) .
the analytical power of bioinformatic platforms combined with patient clinical data from emrs can reveal potential biomarkers and clinical phenotypes that allow researchers to develop experimental strategies using selected patient biospecimens stored in tissue banks . for cancer ,
high - quality biospecimens collected at diagnosis , first relapse , and various treatment stages provide crucial resources for study designs . to enlarge biospecimen collections , patient education regarding the value of specimen donation is vital .
one approach for increasing consent is to offer publically available illustrations and game - like engagements demonstrating how wider sample availability facilitates development of novel therapies .
the critical value of tissue bank samples , bioinformatics , and emr in the early stages of the biomarker discovery process for personalized medicine is often overlooked .
the data obtained also require cross - disciplinary collaborations to translate experimental results into clinical practice and diagnostic and prognostic use in personalized medicine . |
early age at menarche , the onset of menstrual periods in girls , is associated with increased risks of adverse health outcomes such as breast , ovarian , and endometrial cancer , hypertension , type 2 diabetes , and cardiovascular disease [ 1 , 2 ] .
earlier age at menarche is also associated with increased risk for a number of psychosocial outcomes in adolescence including depression , eating disorders , substance abuse , sexual risk - taking and teenage pregnancy .
it has been suggested that childhood bmi has a causal effect on the risk for early menarche and there are a number of strongly plausible biological mechanisms [ 4 , 5 ] .
however , discordant secular trends in obesity and age at menarche have raised doubts about the causal nature of these associations . in developed countries , a long - term trend towards earlier
menarche has been observed from the late 1800s to the mid 1900s . in many countries
these trends appear to have slowed or even stopped since around 1950 while the prevalence of childhood overweight and obesity has increased since the 1980s .
it is possible , therefore , that the apparent association between higher bmi and earlier age at menarche might be confounded by other factors such as diet or exposure to endocrine disruptors .
the association could also be explained by reverse causality as the progression of puberty in girls is accompanied by rapid gains in body weight and body fat .
mendelian randomisation , using robust genetic variants as instrumental variables , has been suggested as an approach to avoid the problems of confounding , residual confounding and specificity that are experienced by traditional epidemiological studies .
for example , mendelian randomisation studies have demonstrated the causal effects of low - density lipoprotein ( ldl ) cholesterol on risk of myocardial infarction , apparent protective effects of high - density lipoprotein ( hdl ) cholesterol on coronary heart disease , and lack of a causal effect of c - reactive protein ( crp ) on ischemic cerebrovascular disease and carotid intimamedia thickness .
similar approaches , using bmi - increasing variants at fto and mc4r have reported apparent causal effects of bmi on hypertension and markers of atherosclerosis . in recent years , large scale genome wide association ( gwa ) studies have identified several common genetic variants that are robustly associated with increased bmi [ 1720 ] .
furthermore , two large studies have reported that some of these genetic variants for higher bmi are also associated with earlier age at menarche [ 21 , 22 ] .
we therefore used a mendelian randomisation approach to assess the likely causal nature of the observed association between higher bmi and risk of early menarche .
the european prospective investigation into cancer and nutrition-(epic- ) norfolk study is a large , predominantly ethnically homogenous , white european population - based cohort study , which is part of a multicentre international study designed to investigate the relationship between diet and chronic disease .
the epic - norfolk study was approved by the norwich local research ethics committee and informed consent was given by all participants .
age at menarche in completed whole years and weight at age 20 years were ascertained by recall in the baseline questionnaire which women completed at age 3977 years old .
weight at age 20 was recalled and adult height measured at baseline by trained nurses .
the measures were used to calculate bmi at age 20 ( recalled weight in kg divided by height squared in metres ) .
genotype information was available for 12 variants in the first 12 loci that were identified in the first three waves of gwa studies for bmi [ 1720 ] ; these variants were rs91121980 ( in / near to gene fto ) ; rs17782313 ( mc4r ) , rs6548238 ( tmem18 ) , rs10938397 ( gnpda2 ) , rs368794 ( kctd15 ) , rs32568958 ( negr1 ) , rs10838738 ( mtch2 ) , rs925946 ( bdnf ) , rs7498665 ( sh2b1 ) , rs10913469 ( sec16b ) , rs10938397 ( faim2/bcdin3d ) and rs7647305 ( etv5 ) .
genotyping was performed by custom taqman snp genotyping assays ( applied biosystems , warrington , uk ) or ( markers rs10938397 and rs10838738 ) sequenom iplex gold standard chemistry ( sequenom , san diego , ca ) as previously described .
call rates were > 95% and each locus genotyped was under hw equilibrium given = 0.05 . in the epic - norfolk study ,
of these , only 6,709 women had complete genotype data on all 12 snps , however a further 3,972 women had genotype data on at least 9 snps and for these women we imputed genotype data on their missing ( up to 3 ) snps using the mean number of bmi - increasing alleles at each snp as the individual values ( we excluded 276 women who lacked genotype data on more than 3 snps ) .
of the 10,681 women with complete or imputed genotype data , 10,136 had data on recalled age at menarche within the physiological range of 818 years , and of these 8,387 women had data on their recalled body weight at age 20 years , and 8,156 also had data on measured height in order to calculate bmi at age 20 .
all analyses were therefore based on 8,156 women , who were slightly younger , taller and lighter at their baseline visit compared to excluded women , but had very similar recalled weight and ( calculated ) bmi at age 20 and recalled age at menarche ( table 1 ) .
the outcome variable early menarche was defined as menarche before age 12 years , as previously described [ 25 , 26 ] . as the association of interest was between prepubertal bmi and age at menarche , bmi at age 20 years
was used as a proxy for prepubertal bmi . the directly observed increase in risk of early menarche per 1 kg / m change in bmi at age 20
was assessed by logistic regression ( to estimate an odds ratio ) , and by binomial regression ( to estimate an absolute increase in risk ) , with adjustment for age at baseline . to represent an instrumental variable ( iv ) for higher bmi , a
bmi - increasing - allele - score was created in epic - norfolk by summing the number of bmi - increasing alleles across all 12 loci in each person .
the association between the bmi - increasing - allele - score and bmi at age 20 was analysed by linear regression , with adjustment for age at baseline .
the association between the bmi - increasing - allele - score and risk of early menarche was analysed by logistic regression .
the iv - predicted risk for early menarche per 1 kg / m change in bmi and was calculated using the ivprobit command in stata with a maximum likelihood estimator in order to calculate the predicted absolute risk probabilities .
associations between the individual variants and bmi at age 20 years in epic - norfolk women are shown in table 2 .
all 12 variants showed directionally consistent associations with bmi at age 20 as expected from the original reports [ 1720 ] .
the bmi - increasing - allele - score ranged from 3 to 20 alleles and was normally distributed ( figure 1 ) . because few women had a score below 6 ( n = 51 ) or above 17 (
n = 25 ) in figure 1 these scores were collapsed into the categories : less than or equal to 6 and greater than or equal to 17 and imputed snp counts were rounded to the nearest whole number for the figure . on average , each additional bmi - increasing allele was associated with 0.12 kg / mhigher bmi at age 20 ( 95% ci : 0.100.15 , p = 6.8 10 ) , but showed no association with adult height ( p = .3 ) .
1,766 ( 21.7% ) epic - norfolk women reported that their menarche occurred before age 12 years ( early menarche ) .
each 1 kg / m higher bmi at age 20 was directly associated with an 11% ( 95% ci : 914% ) higher relative risk of early menarche , or in terms of absolute risk 1.7% higher ( 1.5 to 2.0% ) .
the bmi - increasing - allele - score was positively associated with the relative risk of early menarche ( or = 1.06 per allele , 95% ci = 1.031.08 , p = 5.8 10 ) . when using the bmi - increasing - allele - score as an instrumental variable for bmi , each 1 kg / m increase in bmi at age 20
was predicted to result in a 6.5% ( 95% ci : 4.68.5% ) increase in the absolute risk of early menarche .
we performed a sensitivity analysis by calculating a weighted bmi - increasing - allele - score , where the contribution of each genotype to the score was weighted by its individual association with bmi at age 20 . using this weighted bmi - increasing - allele - score , each 1 kg / m increase in bmi at age 20
was predicted to result in a 6.7% ( 95% ci : 5.08.4% ) higher absolute risk of early menarche .
the results of this mendelian randomisation analysis infers a causal effect of higher bmi on increased risk of early menarche ( at age < 12 years ) , and that the observed association is unlikely to be explained by positive confounding or by reverse causality .
it is not suggested that such genetic instrumental variable approaches can replace randomised controlled trials , but rather that they support the causal inference from observational studies .
firstly , that there is a reliable association between the genetic variant and the exposure , childhood bmi .
while we did not have information on childhood bmi in this study , the effect size of our 12-variant bmi - increasing - allele - score on bmi at 20 years ( + 0.12 kg / m per allele , or around 0.04 of an sd ) is identical to that in 915-year - old children with research clinic measurements in the european youth heart study ( 0.04 sd per allele ; n = 2,042 children ) , and is similar to the effect size of a 10-variant score at age 9 years in the alspac study ( bmi : 0.07 sd per allele ; weight 0.05 sd per allele .
we are therefore confident that our genetic score is a valid instrumental variable for childhood bmi .
secondly , the genetic variants should not have pleiotropic effects on different biological processes , or be in linkage disequilibrium with other genetic variants that might directly affect the outcome .
we would expect that pleiotropic effects ( i.e. , mediated by independent biological processes ) on bmi and timing of menarche are infrequent among bmi variants .
in contrast , a recent very large study in 87,000 women reported that individually 9 of the 12 bmi - increasing variants that we studied here showed significant associations with lower age at menarche .
as most bmi - increasing variants are associated with lower age at menarche , we consider that pleiotropy is unlikely and therefore our findings indicate a causal pathway linking higher bmi , or the growth and developmental processes that lead to higher bmi , to earlier menarche . it is unclear why our iv - predicted association , based on the genetic score , was even stronger than the directly observed association between bmi and early menarche .
the mechanisms of action for these variants are yet unknown and it is possible that some might indeed have direct effects on the timing of menarche .
alternatively , the observed association between childhood bmi and age at menarche might have been artificially diminished due to negative confounding or due to our imprecise estimate of childhood bmi . in this case
, the iv - predicated association may actually be closer to true size of the causal association between childhood bmi and age at menarche .
the lack of direct assessments of growth during childhood was a limitation of our study .
recalled body weight at age 20 years was used to calculate a proxy measure of childhood bmi .
however , other studies have demonstrated that recall of early adult weight in this age group is reliable and that bmi at age 20 is well - correlated with childhood bmi .
furthermore in our iv analyses , bmi at 20 is only used in order to estimate the effect size of the genetic score on childhood bmi , and the result was very similar to that found in earlier childhood studies ( discussed above ) .
our inference of a causal relationship between higher bmi and early menarche is supported by other sources of evidence .
one randomised control trial showed that reduction in childhood obesity led to avoidance of early menarche .
however , the lifestyle intervention used in that trial lead to changes in fruit and vegetable consumption , duration of physical activity and sedentary behaviour , and it is therefore not possible to specify any one causal factor .
most longitudinal studies , while small in numbers , consistently report that higher bmi during childhood is associated with subsequent increased risk of early menarche [ 31 , 3336 ] ( table 3 ) . in those longitudinal studies , associations with earlier menarche were seen with childhood bmi at various ages from 9 months through to 12 years .
one study reported that greater weight gain even in the first weeks and months of life was associated with earlier age at menarche .
further longitudinal studies are needed to identify the relative effects of rapid weight during different childhood ages , up to and including the pubertal years , on the timing of menarche . in a separate birth cohort study we recently examined the relationship between the age at menarche genetic variant in lin28b to measures of growth and weight gain from birth to age 53 years . in that study , that the menarche - lowering allele was associated with higher bmi in women from ages 1543 years , but not before age 15 . that finding is therefore consistent with our current analysis in supporting a causal direction of childhood bmi on puberty timing , although puberty timing may in turn influence bmi beyond puberty .
together , these findings indicate that the occurrence of early menarche is likely to increase as the prevalence of childhood overweight and obesity increases .
an expert panel assembled to assess the evidence for an ongoing secular trend in age at menarche since 1940 failed to reach a consensus , although the majority concluded that there was sufficient evidence for an ongoing decline .
the rapid reductions in mean age at menarche from the late 1800s to the mid 1900s have been attributed to the avoidance of under - nutrition , while the effects of overnutrition on further advancing age at menarche may result in relatively slower yet continuing trends to earlier menarche .
our genetic iv analysis infers a strong causal effect of higher childhood bmi on the risk of early menarche .
while mendelian randomisation analysis is dependent on a number of assumptions , our conclusions are supported by the findings of longitudinal studies and suggest that increasing trends in childhood obesity will lead to increasing prevalence of early menarche .
| to infer the causal association between childhood bmi and age at menarche , we performed a mendelian randomisation analysis using twelve established
bmi - increasing
genetic variants as an instrumental variable ( iv ) for higher bmi . in 8,156 women of european descent from the epic - norfolk cohort ,
height was measured at age 3977 years ; age at menarche was self - recalled , as was body weight at age 20 years , and bmi at 20 was calculated as a proxy for childhood bmi .
dna was genotyped for twelve bmi - associated common variants ( in / near fto , mc4r , tmem18 , gnpda2 , kctd15 , negr1 , bdnf , etv5 , mtch2 , sec16b , faim2 and sh2b1 ) , and for each individual a bmi - increasing - allele - score was calculated by summing the number of bmi - increasing alleles across all 12 loci .
using this bmi - increasing - allele - score as an instrumental variable for bmi , each 1 kg / m2 increase in childhood bmi was predicted to result in a 6.5% ( 95% ci : 4.68.5% ) higher absolute risk of early menarche ( before age 12 years ) . while mendelian randomisation analysis is dependent on a number of assumptions , our findings support a causal effect of bmi on early menarche and suggests that increasing prevalence of childhood obesity will lead to similar trends in the prevalence of early menarche . |
the initiation of coagulation , especially in severe sepsis , is probably mediated by the induction of tissue factor ( tf ) expression by endotoxin .
this leads to the activation of factor x , which then combines with factor va to convert prothrombin to thrombin .
although thrombin is usually considered to have purely procoagulant activity , it is in fact a multifunctional protein that has some important homeostatic anticoagulant effects ( fig .
one of its main functions is to bind to thrombomodulin , which is expressed on the endothelial cell surface .
thrombomodulin is the major physiologic buffer for the procoagulant effects of thrombin in normal vessels . because thrombomodulin binds to the same site on thrombin that would normally bind to fibrinogen , platelets or factor v ,
instead , the thrombin - thrombomodulin complex activates protein c ( through a different site on the thrombin molecule ) , resulting in initiation of the activated protein c pathway .
activated protein c must dissociate from epcr before it can bind to protein s and function as an effective anticoagulant through the inactivation of factor va .
ec , endothelial cell ; paf , platelet - activating factor ; pdgf , platelet - derived growth factor ; pmn , polymorphonuclear leukocyte ; tgf , transforming growth factor
although the number of thrombomodulin molecules per endothelial cell is approximately constant , the local concentration of thrombomodulin is determined by the number of endothelial cells that are in contact with the blood . because the endothelial cell surface area per unit blood volume is much greater in the microcirculation ( approximately 3000 - 5000 cm of endothelium / ml blood ) than in larger blood vessels ( approximately 1.5 cm / ml ) ( fig .
2 ) , there is a correspondingly high concentration of thrombomodulin in the normal microvasculature ( approximately 500 nmol / l ) as compared with the larger vessels ( approximately 0.1 - 0.2 nmol / l ) . as a result , thrombin is rapidly removed from the microcirculation as it is bound to thrombomodulin .
this suggests that the activated protein c system is uniquely poised to regulate coagulopathies in the microcirculation , and this has been confirmed in clinical studies .
importantly , at least in some forms of sepsis , thrombomodulin can be downregulated , with resulting loss of ability to regulate thrombin , and hence clotting , in the microcirculation .
homozygous protein c deficiency has a similar result , such that affected babies develop microvascular thrombosis ( purpura fulminans ) almost immediately after birth .
in addition to its procoagulant and anticoagulant effects , thrombin can also be involved in the process of inflammation ( fig .
, it can activate endothelial cells to express p - selectin , causing neutrophil and monocyte adhesion .
it is also chemotactic for polymorphonuclear leukocytes and is a potent inducer of endothelial platelet - activating factor formation .
platelet - activating factor is a major activator of neutrophils , which have been demonstrated to play an important role in the pathogenesis of sepsis .
the inflammatory response also modulates the coagulation system , including downregulation of the expression of thrombomodulin ( fig .
3 ) . inflammation also increases levels of 1-antitrypsin , which is an acute - phase response protein and an important inhibitor of the protein c pathway .
acute inflammatory mediators such as tumor necrosis factor ( tnf)- and endotoxin amplify the production of tf , particularly by monocytes , triggering further coagulation .
complement activation by endotoxin increases exposure of clot - promoting membrane phospholipids to blood , and thereby propagates the coagulation response . in certain forms of inflammation ,
levels of both fibrinogen and plasminogen activator inhibitor ( pai)-1 ( an inhibitor of fibrinolysis ) are increased .
if there were no mechanisms to disrupt the amplification of coagulation by inflammation , and vice versa , then the ensuing vicious cycle would have a catastrophic effect , leading ultimately to death ( fig .
it was hypothesized , therefore , that endogenous activated protein c interferes with these amplification loops by modulating both coagulation and inflammation , which differentiates it from other types of anticoagulants , and that protein c depletion may occur in severe sepsis . the inflammation - coagulation autoamplification loop . adapted from esmon et al .
a primate model was used in the initial study undertaken to investigate this possibility . in animals infused with a lethal escherichia coli dose alone ,
5a ) , and the animals died within 24 - 32 h. in contrast , organ damage was prevented in animals given a lethal dose of e. coli plus an infusion of activated protein c ( fig .
importantly , it was shown that animals could still be rescued if activated protein c was given 2 - 3 h after the lethal e. coli dose was administered .
similarly , when animals were given a sublethal dose of e. coli and protein c activation was blocked with an antibody , mimicking the clinical situation in a patient with sepsis and low protein c levels , the sublethal bacterial dose became lethal , with complete fibrinogen consumption , a disseminated intravascular coagulation - like syndrome , organ failure , and a full - blown septic shock - like response ( fig .
sgpt , serum glutamic pyruvic transaminase ( indicator of liver damage ) . adapted from taylor et al .
hpc4 , murine monoclonal antibody to human protein c ; ig , immunoglobulin ; sgpt , serum glutamic pyruvic transaminase ( indicator of liver damage ) . adapted from taylor et al .
c4-binding protein ( c4bp ) is an acute - phase protein that is known to bind and inhibit protein s , a vital cofactor for the activated protein c system . in another primate study
, it was shown that administration of c4bp together with a normally sublethal dose of e. coli resulted in complete fibrinogen consumption and death . in the same study , it was shown that , whereas either sublethal e. coli doses or c4bp alone had no appreciable effect on tnf- production , administration of both substances led to a circulating tnf- level that approached that seen with a lethal dose of e. coli ( fig .
7 ) , with rapid consumption of fibrinogen , systemic organ damage , and eventual death . with co - administration of protein
s , however , tnf- levels remained similar to those seen in animals administered a sublethal dose of e. coli alone ( fig .
this suggests that reversal of the inhibitory effects of inflammatory cytokines on the activated protein c pathway can control the inflammatory response .
tnf- production in response to sublethal or lethal e. coli infusion , with or without c4bp and protein s ( ps ) supplementation .
c4bp and ps were administered at doses of 20 mg / kg and 3 mg / kg , respectively .
slec , 10% of a lethal dose of e. coli ; lec , 100% lethal dose dose of e. coli . adapted from taylor et al .
one involves the ability of activated protein c to form a tight complex with pai-1 . once the complex with activated protein c forms , pai-1 ( the major inhibitor of tissue plasminogen activator ) can no longer inhibit tissue plasminogen activator . because of the ability of activated protein c to limit thrombin generation , it can also reduce the activation of a precarboxypeptidase b commonly referred to as thrombin activatable fibrinolysis inhibitor .
thrombin activatable fibrinolysis inhibitor functions by removing lysine residues from the fibrin clot that would normally play a role in augmenting plasminogen activation and the fibrinolytic activity of plasmin .
the potentially protective effect of activated protein c has also been shown in two rat studies , both of which suggested inhibition of leukocyte activation as an important mechanism . in one of these studies ,
activated protein c was shown to reduce motor disturbance resulting from spinal cord injury by reducing local levels of tnf- , thereby inhibiting neutrophil accumulation . in the other study ,
animals treated with activated protein c were found to have a reduction in ischemia / reperfusion - induced renal injury , which was associated with reductions in renal levels of tnf- , interleukin-8 and myeloperoxidase , with resulting inhibition of leukocyte activation .
activated protein c has a number of properties that differentiate it from other anticoagulants , such as heparin or antithrombin .
first , the activated protein c system is geared to modulate coagulopathy within the microvasculature , and it is microvascular disease that lies at the root of sepsis .
second , the activated protein c system involves the epcr , which was identified during research to discover possible receptors and pathways linked to the system .
the epcr was found to be homologous to the major histocompatibility complex class i family of molecules , all of which are involved in the regulation of inflammation .
the epcr is synthesized on the endothelial cell surface and moves to specialized regions ( caveolae ) in the endothelium , through which it is able to enter the cell and then the cell nucleus . once inside the nucleus , it is able to redirect the gene expression profiles within the cell . unlike other plasma membrane receptors ,
epcr is also capable of carrying activated protein c into the nucleus , with subsequent alterations to some of the gene products . activated protein c / epcr - mediated anticoagulant pathway .
apc , activated protein c ; mp , metalloproteinase ; palm , palmitate ; pc , protein c ; pma , phorbol myristate acetate ; tm , thrombomodulin ; rrc , arg , arg , lys . adapted from esmon .
furthermore , when thrombin or inflammatory cytokines come into contact with the endothelium , they induce shedding of epcr from the cell surface , a process that is mediated by a metalloproteinase .
soluble epcr binds to proteinase 3 , a serine proteinase that is found in neutrophils .
this soluble epcr - proteinase 3 complex , either alone or in a complex with protein c , appears to interact with neutrophils via an integrin ( mac-1 ) to inhibit their adhesion to the endothelium , and as a result prevents the migration of the neutrophils into the tissues .
several unique mechanisms by which activated protein c can affect inflammation and coagulation have been demonstrated in various in vitro studies . in one study , it was shown that incubation of monocytes with activated protein c leads to a decrease in tnf- production and blockade of the translocation and activation of the central proinflammatory transcription factor nuclear factor-b in response to lipopolysaccharide exposure .
the binding of activated protein c to a specific receptor on mononuclear phagocytes has also been shown to inhibit intracellular calcium signaling and monocyte - dependent t - cell proliferative responses .
finally , epcr - dependent tf suppression by activated protein c has been demonstrated in a human monoblastic leukemia cell line .
the sepsis cascade , which is associated with simultaneous activation of systemic inflammation and coagulation , along with altered fibrinolysis , leads to microvascular endothelial injury , acute organ dysfunction , and possibly death .
thrombin , with its procoagulant and anticoagulant effects , as well as its involvement in the process of inflammation , is thought to be central to the sepsis cascade . in vitro and animal models
suggest that activated protein c , a protein that has antithrombotic , anti - inflammatory and profibrinolytic activities , may be an important modulator of the vicious cycle of coagulation and inflammation associated with the sepsis cascade , which if unchecked may ultimately lead to death .
several properties of activated protein c , including its unique positioning to regulate coagulopathies in the microvasculature and the ability of epcr to facilitate protein c activation , support the continued development of activated protein c - mediated strategies that are aimed at disrupting the process whereby inflammation initiates coagulation and coagulation amplifies inflammation .
c4bp = c4-binding protein ; epcr = endothelial cell protein c receptor ; pai = plasminogen activator inhibitor ; tf = tissue factor ; tnf = tumor necrosis factor . | thrombin is a multifunctional protein , with procoagulant , inflammatory and anticoagulant effects .
binding of thrombin to thrombomodulin results in activation of protein c and initiation of the activated protein c anticoagulant pathway , a process that is augmented by the endothelial cell protein c receptor ( epcr ) .
activated protein c has demonstrated antithrombotic , anti - inflammatory , and profibrinolytic properties .
its antithrombotic activity is particularly important in the microcirculation , and protein c deficiency is associated with microvascular thrombosis . activated protein c has also been shown to modulate inflammation . when the level of thrombomodulin or protein c is reduced in sepsis
there is a vicious cycle of coagulation and inflammation , with potentially lethal consequences . in vitro studies and animal models
have shown that activated protein c blunts the inflammatory and coagulant response to sepsis through a variety of mechanisms . |
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